No-modified hemoglobins and uses therefore

ABSTRACT

S-nitrosohemoglobin (SNO-Hb) can be formed by reaction of Hb with S-nitrosothiol and by other methods described herein which do not result in oxidation of the heme Fe. Other methods can be used which are not specific only for thiol groups, but which nitrosate Hb more extensively, and may produce polynitrosated metHb as a product or intermediate product of the method. SNO-Hb in its various forms and combinations thereof (oxy, deoxy, met; specifically S-nitrosylated, or nitrosated or nitrated to various extents) can be administered to an animal or human where it is desired to oxygenate, to scavenge free radicals, or to release NO +  groups to tissues. Thiols and/or NO donating agents can also be administered to enhance the transfer of NO +  groups. Examples of conditions to be treated by SNO-Hbs or other nitrosated or nitrated forms of Hb include ischemic injury, hypertension, angina, reperfusion injury and inflammation, and disorders characterized by thrombosis. Further embodiments of the invention are methods for assessing oxygen delivery to the tissues-of a mammal by measuring SNO-Hb and nitrosylhemoglobin in blood.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/796,164 filed on Feb. 6, 1997, which is acontinuation-in-part of PCT/US96/14659 filed on Sep. 13, 1996, which isa continuation of U.S. patent application Ser. No. 08/667,003 filed onJun. 20, 1996, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/616,371 filed on Mar. 15, 1996, which claimspriority to U.S. Provisional Application No. 60/003,801 filed onSeptember 15, 1995. This application is also a continuation-in-part ofPCT/US96/14660 filed on Sep. 13, 1996, which is a continuation of U.S.patent application Ser. No. 08/616,259 filed on Mar. 15, 1996, whichclaims priority to U.S. Provisional Application No. 60/003,801 filed onSep. 15, 1995. The teachings of all of the above applications are eachincorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. NIH RO1HL52529 awarded by the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

Interactions of hemoglobin (Hb) with small diffusible ligands, such asO₂, CO₂ and NO, are known to occur at its metal centers and aminotermini. The O₂/CO₂ delivery functions, which arise in the lung andsystemic microvasculature, are allosterically controlled. Suchresponsiveness to the environment has not been known to apply in thecase of NO. Specifically, it has been thought previously that NO doesnot modify the functional properties of Hb to any physiologicallysignificant degree. Kinetic modeling predicts that the vast majority offree NO in the vasculature should be scavenged by Hb (Lancaster 1994).Accordingly, the steady-state level of NO may fall below the K_(m) fortarget enzymes such as guanylate cyclase (Lancaster 1994), if not in theunperturbed organism, then with oxidant stress such as that found inatherosclerosis. These considerations raise the fundamental question ofhow NO exerts its biological activity.

One answer to this question is found in the propensity of nitric oxideto form S-nitrosothiols (RSNOs) (Gaston, B. et al., Proc. Natl. Acad.Sci. USA 90:10957-10961 (1993)), which retain NO-like vasorelaxantactivity (Stamler, J. S., et al., Proc. Natl. Acad. Sci. USA 89:444-448(1992)), but which can diffuse freely in and out of cells, unlike Hb. Inparticular, the NO group of RSNOs possesses nitrosonium (NO⁺) characterthat distinguishes it from NO itself. It is increasingly appreciatedthat RSNOs have the capacity to elicit certain functions that NO isincapable of (DeGroote, M. A. et al., Proc. Natl. Acad. Sci. USA92:6399-6403 (1995); Stamler, J. S., Cell 78:931-936 (1994)). Moreover,consideration has been given to the possibility that —SNO groups inproteins serve a signaling function, perhaps analogous tophosphorylation (Stamler, J. S. et al., Proc. Natl. Acad. Sci. USA89:444-448 (1992); Stamler, J. S. Cell, 78:931-926 (1994)). AlthoughS-nitrosylation of proteins can regulate protein function (Stamler, J.S. et al., Proc. Natl. Acad. Sci. USA 89:444-448 (1992); Stamler, J. S.,Cell, 78:931-936 (1994)), intracellular S-nitrosoproteins —the sine quanon of a regulatory posttranslational modification—has heretofore notbeen demonstrated.

Hemoglobin is a tetramer composed of two alpha and two beta subunits. Inhuman Hb, each subunit contains one heme, while the beta (β) subunitsalso contain highly reactive SH groups (cysβ93) (Olson, J. S., Methodsin Enzymology 76:631-651 (1981); Antonini, E. & Brunori, M. InHemoglobin and Myoglobin in Their Reactions with Ligands, AmericanElsevier Publishing Co., Inc., New York, pp. 29-31 (1971)). Thesecysteine residues are highly conserved among species although theirfunction has remained elusive.

NO (nitric oxide) is a biological “messenger molecule” which decreasesblood pressure and inhibits platelet function, among other functions. NOfreely diffuses from endothelium to vascular smooth muscle and plateletand across neuronal synapses to evoke biological responses. Under someconditions, reactions of NO with other components present in cells andin serum can generate toxic intermediates and products at localconcentrations in tissues which are effective at inhibiting the growthof infectious organisms. Thus, it can be seen that a method ofadministering an effective concentration of NO or biologically activeforms thereof would be beneficial in certain medical disorders.

Platelet activation is an essential component of blood coagulation andthrombotic diathesis. Activation of platelets is also seen inhematologic disorders such as sickle cell disease, in which localthrombosis is thought to be central to the painful crisis. Inhibition ofplatelet aggregation is therefore an important therapeutic goal in heartattacks, stroke, peripheral vascular disease and shock (disseminatedintravascular coagulation). Researchers have attempted to giveartificial hemoglobins to enhance oxygen delivery in all of the abovedisease states. However, as recently pointed out by Olsen and coworkers,administration of underivatized hemoglobin leads to platelet activationat sites of vascular injury (Olsen S. B. et al., Circulation 93:327-332(1996)). This major problem has led experts to conclude that cell-freeunderivatized hemoglobins pose a significant risk of causing blood clotsin the patient with vascular disease or a clotting disorder (Marcus, A.J. and J. B. Broekman, Circulation 93:208-209 (1996)). New methods ofproviding for an oxygen carrier and/or a method of inhibiting plateletactivation would be of benefit to patients with vascular disease or whoare otherwise at risk for thrombosis.

SUMMARY OF THE INVENTION

The invention relates to methods of forming SNO-Hb (S-nitrosohemoglobin,which includes for instance, oxy-, deoxy-, or met-hemoglobin) byreaction of Hb with S-nitrosothiol in procedures which avoid oxidationof the heme. The invention also includes methods of producing nitrosated(including nitrosylated at thiols or metals) and nitrated derivatives ofhemoglobins in which the heme Fe can be oxidized or not oxidized,depending on the steps of the method. The invention also relates to amethod of therapy for a condition in which it is desired to oxygenate,to scavenge free radicals, or to release NO⁺ groups or other forms ofbiologically active NO to tissues. A composition comprising SNO-Hb inits various forms and combinations thereof (oxy, deoxy, met;specifically S-nitrosylated, or nitrosated or nitrated to variousextents) can be administered to an animal or human in these methods.Compositions comprising thiols and/or NO donating agents can also beadministered to enhance the transfer of NO⁺ groups. Examples ofconditions to be treated by nitrosated or nitrated forms of hemoglobininclude ischemic injury, hypertension, angina, reperfusion injury andinflammation, and diseases characterized by thrombosis. Furtherembodiments of the invention are methods for assessing oxygen deliveryto the tissues of a mammal by measuring SNO-Hb and nitrosylhemoglobin inblood.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIGS. 1A-1D are spectrographs of different forms of Hb as described inExample 1.

FIG. 2A is a graph showing formation, with time, of SNO-Hb byS-nitrosylation.

FIG. 2B is a graph showing the decomposition, with time, of oxy anddeoxy forms of SNO-Hb.

FIG. 3A is a graph showing the loading of red blood cells (erythrocytes)with S-nitrosocysteine, over time. The inset is a series ofspectrographs of forms of Hb as described in Example 3.

FIG. 3B is a series of tracings recording isometric tone of a rabbitaortic ring following treatment of the aortic ring with various agentsas described in Example 3.

FIG. 4A is a graph of change in tension of a rabbit aortic ring versusconcentration of the Hb used in the experiment.

FIG. 4B is a graph of change in tension of a rabbit aortic ring versusconcentration of the Hb used in the experiment, where glutathione wasalso added to test the effect as compared to FIG. 4A.

FIG. 4C is a graph of the ratio of S-nitrosoglutathione formed/startingSNO-Hb concentration versus time, showing rates of NO group transferfrom oxy and met forms of Hb to glutiathione.

FIG. 4D is a graph of S-nitrosothiols exported from loaded red bloodcells over time.

FIG. 5 is a graph showing the mean arterial blood pressure in rats afterthey received various doses of oxyHb (▴), SNO-oxyHb (▪), or SNO-metHb(●).

FIGS. 6A-6F are a series of tracings recording blood pressure (FIGS. 6Aand 6B), coronary artery diameter (FIGS. 6C and 6D) and coronary arteryflow (FIGS. 6E and 6F), after administration of S-nitrosohemoglobin toanesthetized dogs.

FIG. 7A is a graph illustrating the effect of unmodified HbA₀ onplatelet aggregation. The maximal extent of aggregation of platelets isplotted against the concentration of HbA (10 nM to 100 μm) preincubatedwith platelets. Experiments were performed as in Example 9. Verticalbars plotted with each data point indicate the standard deviation.

FIG. 7B is a graph illustrating the effect of S-nitroso(oxy)hemoglobinon platelet aggregation. The normalized maximal extent of aggregation ofplatelets is plotted against the concentration of HbA (10 nM to 100 μm)preincubated with platelets.

FIG. 7C is a graph illustrating the antiaggregation effects on plateletsby S-nitroso(met)hemoglobin.

FIG. 8 is a bar graph showing the amount of cGMP (guanosine 3′,5′-cyclicphosphoric acid), assayed as in Example 10, for 1, 10 and 100 μMconcentrations of native Hb, SNO-oxyHb or SNO-metHb interacting with 10⁸platelets.

FIG. 9A is a graph which shows the spectra (absorbance versus wavelengthin nanometers) of HbA₀ treated as described in Example 11. The shift inthe wavelength of maximum absorbance of spectrum B relative to spectrumA illustrates the extent of addition of NO groups to HbA₀.

FIG. 9B is a graph which shows the spectra of Hb treated with 100-foldexcess S-nitrosoglutathione as described in Example 11.

FIG. 9C is a graph which shows the spectra of HbA₀ treated with excessS-nitrosocysteine as described in Example 11.

FIG. 9D is a graph which shows the spectra of rat Hb treated with100-fold excess S-nitrosocysteine. Spectrum A shows nitrosated Hb notfurther treated with dithionite; spectrum B shows nitrosated Hb furthertreated with dithionite.

FIG. 9E is a graph illustrating the increase in nitrosated Hb productwith time by reacting HbA₀ with either 100×excess S-nitrosocysteine (topcurve) or 10×excess S-nitrosocysteine (middle curve). HbA₀ waspreincubated with 100 μM inositol hexaphosphate before reacting with10×excess S-nitrosocysteine (bottom curve; triangle points). (SeeExample 11.)

FIG. 10 is a graph illustrating the percent change, with time, in bloodflow measured in caudatoputamen nucleus of rats after injection of therats with: ◯, 100 nmol/kg SNO-Hb; ●, 1000 nmol/kg SNO-Hb; or ▪, 1000nmol/kg underivatized Hb (see Example 12).

FIG. 11 is a graph illustrating the percent change in tension of a ringof aorta from rabbit, plotted as a function of the log of the molarconcentration of hemoglobin tested (see Example 13). ●, Hb treated withS-nitrosocysteine at a ratio of 1:1 CYSNO/Hb; ◯, Hb treated with CYSNOat a ratio of 10:1 CYSNO/Hb; ♦, Hb treated with CYSNO at a ratio of100:1.

FIG. 12 is a graph of the absorbance versus the wavelength of light(nm), for aqueous solutions of 17 μM deoxyhemoglobin, 1 μM NO, andvarying amounts of dissolved oxygen added by sequential injections ofroom air. The absorbance of the initial solution (no added air) is shownby the curve with the highest peak at approximately 430 nm. Sequentialadditions of 50 μl of air shift the curve leftwards on the graph. SeeExample 14.

FIG. 13 is a graph showing the yield of SNO-Hb as micromolarconcentration (left axis, diamonds) and as % of NO added (right axis,squares), plotted against the heme:NO ratio, when nitrosyl-deoxyHb madeat various ratios of heme:NO was exposed to oxygen. See Example 15.

FIG. 14A is a graph showing difference spectra (each a spectrum of theNo and Hb mixture minus spectrum of the starting deoxyHb), for 17 μMhemoglobin and NO mixtures, for the concentrations of NO shown. SeeExample 16.

FIG. 14B is a graph showing the peak wavelength of the differencespectra plotted against the concentration of nitric oxide added to thesolution as in FIG. 14B.

FIG. 15A is a graph showing difference spectra (deoxyhemoglobin and airmixtures minus initial deoxyhemoglobin spectrum), for successiveadditions of air.

FIG. 15B is a graph showing difference spectra (20 μM deoxyhemoglobinand 1 μM NO mixture, with successive additions of air, minus initialdeoxyhemoglobin spectrum). See Example 17.

FIG. 16 is a graph showing two difference spectra (A₄₁₆ of hemoglobinand No solution at heme:NO 20:1 minus initial deoxyhemoglobin A₄₁₈) forthe mutant β93Ala Hb and wild type β93Cys Hb. See Example 18.

FIG. 17 is a graph showing the yield of SNO-Hb as micromolarconcentration (left axis, diamonds) and as % of NO added (right axis,squares), plotted against the heme:NO ratio, when nitrosyl-deoxyHb madeat various ratios of heme:NO was exposed to oxygen. See Example 19.

FIG. 18A is a graph showing the percentage content of oxidizedhemoglobin (metHb) for different concentrations of Hb (symbols below) towhich NO was added to reach varying final concentrations (horizontalaxis). ♦ represents 1.26 μM hemoglobin, ▪ represents 5.6 μM hemoglobin,▴ represents 7.0 μM hemoglobin, X represents 10.3 μM hemoglobin,represents 13.3 μM hemoglobin, and ● represents 18.3 μM hemoglobin. SeeExample 20.

FIG. 18B is a graph showing the yield of oxidized hemoglobin (μM)plotted against the final concentration of NO added to solutions of Hbat the concentrations indicated by the symbols as for FIG. 18A.

FIG. 19 is a graph showing the concentration of oxidized Hb (metHb)plotted against the NO concentration, in experiments performed asdescribed in Example 21 in 10 mM (♦), 100 mM (Δ), or 1 M () sodiumphosphate buffer, pH 7.4.

FIGS. 20A and 20B are graphs showing the contractile effects of oxyHb,SNO-oxyHb, deoxy-Hb and SNO-deoxy-Hb on thoracic aortic ring isolatedfrom rabbit. Measurements are percent increase in tension of aortic ringas a function of the log of the concentration of hemoglobin orSNO-hemoglobin. Measurements are made after the tension has stabilized.

FIG. 20C is a graph showing the percent change in tension of contractedaortic ring as a function of the log concentration of SNO-hemoglobin atthe concentrations of O₂ indicated, in addition to 10 μM glutathione.

FIG. 20D is a graph showing the percent change in tension of contractedaortic ring as a function of the log concentration of SNO-glutathione,in the concentrations of O₂ indicated.

FIG. 21A and FIG. 21B are each a series of four graphs illustrating thechange with time in tension of rabbit aortic ring upon the addition ofred blood cells treated with S-nitrosocysteine (“red blood cells loadedwith nitric oxide”), or untreated red blood cells, as indicated, in theconcentration of O₂ indicated. FIG. 21C is a graph illustrating thechange with time in tension of rabbit aortic ring contracted withphenylephrine under hypoxic conditions (6-7 torr) and then exposed toeither 1 μM Hb or SNO-Hb.

FIG. 22 is a bar graph depicting the concentrations of FeNO/Hb andSNO/Hb in venous or arterial blood as measured in Example 24.ATA=atmospheres of absolute pressure.

FIGS. 23A-23I are each a graph showing the effects of SNO-Hb (●) and Hb(▪) (1 μmol/kg infused over 3 minutes) on local blood flow in substantianigra (SN), caudate putamen nucleus, and parietal cortex of rats, in 21%O₂ (FIGS. 23A, 23B and 23C), in 100% O₂ (FIGS. 23D, 23E and 23F), and in100% O₂ at 3 atmospheres absolute pressure (FIGS. 23G, 23H and 23I) asmeasured in Example 25.

FIG. 24A is a bar graph showing the percent change in blood pressure ofrats, during exposure to three different conditions (inspired O₂concentrations of 21%, 100%, or 100% O₂ at 3 ATA) upon infusion of GSNO,SNO-Hb, or Hb, as tested in Example 26.

FIG. 24B is a bar graph showing the percent change in blood pressure ofrats [pre-administered (+L-NMMA), or not preadministered (−L-NMMA),N^(G)-monomethyl-L-arginine] upon infusion of SNO-RBCs, as tested inExample 26.

DETAILED DESCRIPTION OF THE INVENTION

Roles for Hemoglobin in Physiology

The increase in SNO-Hb content of red cells across the pulmonary circuit(right ventricular inport-left ventricle) suggests that the Hb moleculeis S-nitrosylated in the lung. Selective transfer of the NO group fromendogenous RSNOs found in lung (Gaston, et al. (1993)) and blood(Scharfstein, J. S. et al., J. Clin. Invest. 94:1432-1439 (1995)) to SHgroups of Hb, substantiate these findings. The corresponding decline inHb(FeII)NO levels across the pulmonary bed reveals a role for the lungeither in the elimination of NO or in its intramolecular transfer fromheme to cysβ93. Taken in aggregate, these data extend the list offunction-regulating interactions of Hb with small molecules within therespiratory system, previously known to include the elimination of COand CO₂, and uptake of O₂. Since, as demonstrated herein, oxygenation ofHb leads to structural changes that increase the NO-related reactivityof cysβ93, O₂ can now be regarded as an allosteric effector of HbS-nitrosylation.

The arterial-venous difference in SNO-Hb concentration suggests that theprotein acts as an NO group donor in the systemic circulation. There isgood indication that SNO-Hb functions in regulation of vasomotor tone.In the microcirculation, where control of blood pressure is achieved,erythrocytes come in intimate contact with endothelial surfaces. Underthese conditions, Hb can contract the vasculature by sharply decreasingthe steady state level of free NO (Lancaster, J. R., (1994)). This isbelieved to contribute to the increases in blood pressure that occurwith infusion of cell-free Hbs (Vogel, W. M., et al., Am. J. Physiol.,251:H413-H420 (1986); Olsen, S. B., et al., Circulation 93:329-332(1996)). The transient nature of such hypertensive responses, however,is consistent with the subsequent formation of SNO-Hb which counteractsthis effect, evidenced by its lowering of blood pressure at naturallyoccurring concentrations. Thus, the capacity of the erythrocyte tosupport the synthesis and metabolism of SNO-Hb is important for normalblood flow.

Mammals must have adopted unique molecular mechanisms to ensure adequateNO delivery in the microcirculation. Results herein suggest that Hb hasevolved both electronic and conformational switching mechanisms toachieve NO homeostasis. Specifically, NO scavenging by the metalcenter(s) of SNO-Hb(FeII)O₂ is sensed through its conversion tomet(FeIII) (FIG. 1B). This electronic switch effects decomposition ofSNO-Hb with NO group release (FIGS. 3A, 3B, 4A). In this manner, theNO-related activity of SNO-Hb is partly determined by the amount of NOscavenged. Changes in O₂ tension also function to regulate NO delivery,as it is observed herein that NO release is facilitated bydeoxygenation. This allosteric effect promotes the efficient utilizationof O₂, as NO controls mitochondrial respiration (Shen, W., et al.,Circulation 92:3505-3512 (1995)).

S-nitrosothiol groups in proteins have been implicated in NO metabolismand in regulation of cellular functions (Stamler, J. S., et al., Proc.Natl. Acad. Sci. USA 89:444-448 (1992); Stamler, J. S., Cell 78:931-936(1994)). The identification of SNO-Hb in erythrocytes is the firstdemonstration of an intracellular S-nitrosoprotein and gives furthercredence to the role of such proteins in cellular regulation. Thequestion arises as to how SNO-Hb relaxes blood vessels when any free NOreleased would be scavenged instantaneously by Hb itself according toprevious theories (Lancaster, J. R., (1994)). Noteworthy in this regardare studies showing that RSNO activity involves nitrosyl (NO⁺) transferto thiol acceptors (Scharfstein, J. S., et al., (1994); Arnelle, D. R.and Stamler, J. S., Arch. Biochem. Biophys. 318:279-285 (1995); Stamler,J. S., et al., Proc. Natl. Acad. Sci. USA 89:7674-7677 (1992)), whichserve to protect the NO-related activity from inactivation at metalcenters. Findings presented herein indicate that S-nitrosothiol/thiolexchange with glutathione (forming GSNO) occurs within erythrocytes, andis influenced by the oxidation state of heme and its occupation byligand. Certain activities of GSNO in bacteria require transport ofintact dipeptide (i.e., S-nitrosocysteinylglycine) across the cellmembrane (DeGroote, M. A., et al., Proc. Natl. Acad. Sci. USA92:6399-6403 (1995)). The data presented below in the Examples show thatS-nitrosothiol transport occurs also in eukaryotic cells. GSNO, orrelated thiol carriers exported by erythrocytes (Kondo, T., et al.,Methods in Enzymology, Packer, L., ed., Academic Press, 252:72-83(1995)), might also initiate signaling in or at the plasmalemma(Stamler, J. S., Cell 78:931-936 (1994)), given reports ofthiol-dependent activation of potassium channels by EDRF (Bolotina, V.M., et al., Nature 368:850-853 (1994)). Alternative possibilities alsomerit consideration. In particular, reports that Hb associates with redcell membranes via cysβ93 (Salhany, J. M. and Gaines, K. C., Trends inBiochem. Sci., pp. 13-15 (January 1981)) places Hb in a position todonate the NO group directly to contacting endothelial surfaces, perhapsvia SNO/SH exchange. Cell surface interactions appear to be operative insignaling mediated by other S-nitrosoproteins (Stamler, J. S., et al.,Proc. Natl. Acad. Sci. USA, 89:444-448 (1992); Stamler, J. S., Cell,78:931-936 (1994)).

The highly conserved Cysβ93 residues in Hb influence the oxygen affinityand redox potential of the heme iron and its physiochemical properties(Garel, C., et al., Biochem. 123:513-519 (1982); Jocelyn, P. C., et al.,Biochemistry of the SH Group, p. 243, Academic Press, London; (1972);Craescu, C. T., J. Biol. Chem. 261:14710-14716 (1986); Mansouri, A.,Biochem. Biophys. Res. Commun., 89:441-447 (1979)). Nonetheless, theirlong sought-after physiological function has remained a mystery. Thestudies herein suggest new sensory and regulatory roles for Hb, in whichCysβ93 functions in transducing NO-related signals to the vessel wall.In particular, the physiological function of Cysβ93, which is invariantin all mammals and birds, is to deliver under allosteric control,NO-related biological activity that cannot be scavenged by heme. Thus,these data bring to light a dynamic circuit for the No group in whichintraerythrocytic Hb participates as both a sink and a donor, dependingon its microenvironment. Such observations provide answers to paradoxesthat arise from conceptual frameworks based solely on diffusional spreadand reaction of free NO (Lancaster, J. R., (1994); Wood and Garthwaite,J. Neuropharmacology 33:1235-1244 (1994)); and has implications thatextend to other thiol- and metal-containing (heme) proteins, such asnitric oxide synthase and guanylate cyclase.

The discoveries reported here have direct therapeutic implications.Specifically, concerns over loss of NO-related activity due toinactivation by blood Hb (Lancaster, J. R., (1994)) are obviated by thepresence of an RSNO subject to allosteric control. SNO-Hb is free of theadverse hypertensive properties of cell-free Hb preparations that resultfrom NO scavenging at the metal centers. A composition comprising one ormore of the various forms of cell-free SNO-Hb (e.g., SNO-Hb[FeII] O₂,SNO-Hb[FeIII], SNO-Hb[FeII] CO) can be administered in apharmaceutically acceptable vehicle to a human or other mammal to act asa blood substitute.

Blood Flow Regulation by S-Nitrosohemoglobin is Controlled by thePhysiological Oxygen Gradient

In the classical allosteric model, Hb exists in two alternativestructures, named R (for relaxed, high O₂ affinity) and T (for tense,low O₂ affinity). The rapid transit time of blood through thecapillaries requires that Hb assume the T-structure to efficientlydeliver O₂ (M. F. Perutz, pp. 127-178 in Molecular Basis of BloodDiseases, G. Stammatayanopoulos, Ed. (W. B. Saunders Co., Philadelphia,1987); Voet, D. and Voet, J. G., pp. 215-235 (John Wiley & Sons Inc.,New York, 1995). The switch from R to T in RBCs normally takes placewhen the second molecule of O₂ is liberated. This allosteric transitionalso controls the reactivity of two highly conserved cysteineβ93residues that can react with ‘NO’. Thiol affinity for NO is high in theR-structure and low in T. This means that the NO group is released fromthiols of Hb in low PO₂ and explains the arterial-venous (A-V)difference in the S-nitrosohemoglobin (SNO-Hb) level of blood (see Table2, Example 8). A major function of (S)NO in the vasculature is toregulate blood flow, which is controlled by the resistance arterioles(Guyton, A. C., in Textbook of Medical Physiology (W. B. Saunders Co.,Philadelphia, 1981) pp. 504-513). It is shown from the Examples hereinthat (partial) deoxygenation of SNO-Hb in these vessels (Duling, B. andBerne, R. M. Circulation Research, 27:669 (1970); Popel, A. S., et al.,(erratum Am. J. Physiol. 26(3) pt. 2). Am. J. Physiol. 256, H921 (1989);Swain, D. P. and Pittman, R. N. Am. J. Physiol. 256, H247-H255 (1989);Torres, I. et al., Microvasc. Res., 51:202-212 (1996); Buerk, D. et al.,Microvasc. Res., 45:134-148 (1993)) actually promotes O₂ delivery byliberating (S)NO. That is, the allosteric transition in Hb functions torelease (S)NO in order to increase blood flow.

O₂ delivery to tissues is a function of the O₂ content of blood andblood flow (Dewhirst, M. W. et al., Cancer Res., 54:3333-3336 (1994);Kerger, H. et al., Am. J. Physiol., 268:H802-H810 (1995)). Blood oxygencontent is largely determined by Hb, which undergoes allosterictransitions in the lung and systemic microvasculature that promote thebinding and release of O₂ (L. Stryer, in Biochemistry L. Stryer, Ed. (W.H. Freeman & Co., San Francisco, 1981) pp. 43-82; Guyton, A. C. inTextbook of Medical Physiology (W. B. Saunders Co., Philadelphia, 1981);Perutz, M. F., pp. 127-178 in Molecular Basis of Blood Diseases, G.Stammatayanopoulos, Ed. (W. B. Saunders Co., Philadelphia, 1987); Voet,D. and Voet, J. G. (John Wiley & Sons Inc., New York, 1995) pp. 215-235pp. 208-215, 224-225, 230-245, 344-355)). Intimate contact betweenerythrocyte and endothelium is believed to facilitate O₂ delivery byminimizing the distance for O₂ diffusion into surrounding tissues (Caro,C. G. et al., Oxford University Press, Oxford, 363 (1978)). On the otherhand, regional blood flow is regulated by metabolic requirements of thetissue: blood flow is increased by hypoxia and decreased when O₂ supplyexceeds demand (Guyton, A. C., in Textbook of Medical Physiology (W. B.Saunders Co., Philadelphia, 1981) pp. 504-513)). These classicalphysiological responses are thought to be partly mediated by changes inthe level of endothelial-derived NO and its biological equivalents(Park, K. H. et al., Circ. Res, 71:992-1001 (1992); Hampl, V. et al., J.Appl. Physiol. 75(4):1748-1757 (1993)).

This standard picture has its problems. First, it is puzzling thatsignificant O₂ exchange occurs in the precapillary resistance vessels(evidenced by the periarteriolar O₂ gradient; Duling, B. and Berne, R.M. Circulation Research, 27:669 (1970); Popel, A. S., et al., (erratumAm. J. Physiol. 26(3) pt. 2). Am. J. Physiol. 256, H921 (1989); Swain,D. P. and Pittman, R. N. Am. J. Physiol. 256, H247-H255 (1989); Torres,I. et al., Microvasc. Res., 51:202-212 (1996); Buerk, D. et al.,Microvasc. Res., 45:134-148 (1993)). Why is O₂ lost to counter-currentvenous exchange prior to reaching the tissues? Second, close contactbetween endothelial surfaces and erythrocytes leads to sequestration ofNO by Hb (Stamler, J. S., Nature, 380:108-111 (1996); Perutz, M. F.,Nature, 380:205-206 (1996)). Decreases in the steady-state levels of NOin terminal arterioles (King, C. E. et al., J. Appl. Physiol.,76(3):1166-1171 (1994); Shen, W. et al., Circulation, 92:3505-3512(1995); Kobzik, L. et al., Biochem. Biophys. Res. Comm., 211(2):375-381(1995); Persson, M. G., et al., Br. J. Pharmacol., 100:463-466 (1990)and capillaries (Mitchell, D., and Tyml, K., Am. J. Physiol., 270 HeartCirc. Physiol., 39), H1696-H1703 (1996)) contract blood vessels, blunthypoxic vasodilation and reduce red cell velocity. This line ofreasoning leads to the paradox: the red blood cell seems to oppose itsown O₂ delivery function (note in vivo effects of Hb in FIGS. 10A-10I).

The finding that the O₂ gradient in precapillary resistance vesselspromotes NO group release from SNO-Hb appears to solve these problems.SNO-Hb compensates for NO scavenging at the heme iron by assuming theT-structure which liberates SNO. Specifically, Cys93 donates the NOgroup in deoxy structure whereas it cannot do so in the oxyconformation. Accordingly, the O₂ gradient determines whether SNO-Hbdilates or constricts blood vessels. Stated another way, SNO-Hb sensesthe tissue PO₂ (i.e., the periarteriolar O₂ gradient) and then utilizesthe allosteric transition as a means to control arteriolar tone. If thetissue is hypoxic (i.e., the O₂ gradient is high), SNO is released toimprove blood flow. However, if O₂ supply exceeds demand (i.e., the O₂gradient is small), SNO-Hb holds on to the NO group by maintaining theR-structure —with the net effect of reducing blood flow in line withdemand. SNO-Hb thereby contributes to the classical physiologicalresponses of hypoxic vasodilation and hyperoxic vasoconstriction.

Based on studies described herein, especially Examples 22-26, thefollowing picture emerges. Partially nitrosylated Hb (Hb[FeII]NO) entersthe lung in T-structure (see venous measurements in FIG. 22). There,S-nitrosylation is facilitated by the O₂-induced conformational changein Hb. SNO-oxyHb (SNO-Hb[FeII]O₂) enters the systemic circulation inR-structure (see arterial levels in FIG. 22). Oxygen losses inprecapillary resistance vessels then effect an allosteric transition(from R to T) in Hb which liberates ‘NO’ to dilate blood vessels (seeespecially FIGS. 20D and 23A-C). NO released from Hb is transferreddirectly to the endothelium, or by way of low mass S-nitrosothiols—suchas GSNO—which are exported from RBCs (see FIG. 4D and Example 4; seealso FIGS. 20C and 24A). Thus, the O₂ gradient in arterioles serves toenhance O₂ delivery: it promotes an allosteric transition in Hb whichreleases NO-related activity to improve blood flow.

Assay Methods

The invention also relates to a method for determining the concentrationof nitrosyl(FeII)—hemoglobin in a blood sample, thereby serving as ameasure of the level of NO in the animal or human from which the bloodsample has been taken. The method is related to one used previously forthe measurement of S-nitrosoproteins and smaller molecular weightS-nitrosothiols in plasma (See U.S. Pat. No. 5,459,076; Oct. 17, 1995.The contents of this patent are hereby incorporated by reference intheir entirety.) However, the primary focus of the present invention ison assaying for nitrosyl(FeII)—hemoglobin rather than S-nitrosothiols.

In contrast to the previous method, in which the red blood cells wereremoved and discarded from the sample to be analyzed, the subjectinvention method uses the red blood cells. The method measures NO whichhas reacted with the thiol groups of hemoglobin in the form ofS-nitroso-hemoglobin (SNO-Hb) as well as No bound to the Fe of the heme(nitrosyl(FeII)-hemoglobin or Hb(FeII)NO). As shown in the table, thelevel of S-nitroso-hemoglobin in venous blood is negligible compared tothe level of Hb(FeII)NO. Therefore, to specifically measure the level ofHb(FeII)NO in venous blood, it is unnecessary to include steps in whichHb samples are divided into two aliquots which are then either treatedor not treated with a 10-fold excess of HgCl₂ over the proteinconcentration. Reaction of Hb with HgCl₂ removes NO from thiol groupsselectively, without disturbing No bound at the heme. Values for NOobtained from the HgCl₂ reaction, if significant, should be subtractedfrom the total NO obtained for the measurements without the HgCl₂reaction, to obtain an accurate value for Hb(FeII)NO.

In one embodiment of the invention, a blood sample is taken from amammal, such as a human, and the solid parts including cells areisolated away from the remaining fluid. The cells are then lysed bystandard methods, and a protein fraction is prepared. Beforequantitating nitric oxide adducts (nitrosonium adducts, which includelow molecular weight S-nitrosothiols such as S-nitrosoglutathione andhigh molecular weight S-nitrosothiols such as S-nitroso-proteins), it ispreferable to first remove low molecular weight S-nitrosothiolsendogenous to the red blood cells, which would also contribute to the NOvalue, by a step which separates low molecular weight molecules awayfrom the red blood cell proteins (referred to as desalting). This stepcan include, for example, dialysis or column chromatography based onseparation by size of the molecules. A further step is to subject theprotein fraction to photolysis, as in a photolysis cell, where it isirradiated with light of the appropriate wavelength to liberate NO fromthe various forms of hemoglobin. The resulting NO is detected byreaction with ozone.

One embodiment of the invention utilizes a chemiluminescence apparatusin which a photolysis cell is linked directly to the reaction chamberand detector portion, thereby bypassing the pyrolyzer. A sample of theblood protein fraction is injected into the photolysis cell, eitherdirectly, or as chromatographic effluent from a high-performance liquidor gas chromatography system which is connected to the photolysis cell.

The sample is then irradiated with a mercury vapor lamp, and directedthrough a series of cold traps, where liquid and gaseous fractions whichare less volatile than nitric oxide (such as nitrite and nitrate) areeliminated, leaving only free nitric oxide remaining in the cell. Thenitric oxide is then transported by a gaseous stream, preferably helium,into the chemiluminescence spectrometer. In the alternative, other inertgases may be used.

Once present in the chemiluminescence spectrometer, the free nitricoxide is detected by its chemical reaction with ozone, resulting in thegeneration of signals that are recorded on a digital integrator. Ifdesired, flow rates and illumination levels in the photolysis cell canbe adjusted to cause complete photolysis of the S-nitric oxide bond ofthe S-nitrosothiol compounds. Flow rates and illumination levels may beadjusted by routine methods available in the art, in order to achieveoptimal cleavage of the bond between the particular adduct and nitricoxide, nitrosonium or nitroxyl, whichever is bound.

In a variation, the invention relates to a method for detectingS-nitrosothiols, including primarily S-nitroso-hemoglobin (SNO-Hb) in ablood sample. This method comprises inactivating the chemiluminescence,signal-generating capability of any nitric oxide which is associatedwith a thiol, in the protein fraction derived from the blood sample, anddetermining the amount of thiol-bound nitric oxide by measuring thequantitative difference between total nitric oxide and nitric oxideremaining after inactivation.

A particular embodiment of this variation relates to a method in whichthe protein fraction derived from the blood sample is treated with asource of mercury ions, mercuric ions being preferred, followed by airincubation, which oxidizes the nitric oxide and nitrosonium and rendersthem undetectable. Compounds suitable for pretreatment include Hg₂Cl₂and other mercurous ion salts and organic mercurials. The treated sampleis then injected into the photolysis cell, where NO⁺ is converted to NO⁺(nitric oxide) and the nitric oxide is detected by the chemiluminescencemethod described above. The amount of nitric oxide which is specificallyderived from S-nitrosothiols is determined by comparing thechemiluminescence signal generated by the mercury ion-treated sample,with a chemiluminescence signal generated by a sample of the equivalentbiological fluid which is not treated with mercury ion prior toinjection into the photolysis cell.

In a further embodiment of the claimed invention, the methods describedherein can be utilized to determine the presence of a disease statewhich involves abnormal levels of nitric oxide and its biologicallyactive equivalents, by monitoring Hb(FeII)NO and SNO-Hb levels in blood,and more particularly, Hb(FeII)NO in venous blood from a patient. Theability to specifically assay for Hb(FeII)NO in venous blooddistinguishes this assay over previously known methods. Nitric oxideadducts represent a pool of bioactive nitric oxide in physiologicalsystems. Therefore, in disease states in which the pathogenesis derivesfrom the effects of abnormal levels of nitric oxide, these methodsprovide a means for the clinician to determine the presence of, andmonitor the extent of, the disease state. Such information enables theclinician to determine the appropriate pharmacological interventionnecessary to treat the disease state. Such disease states and medicaldisorders include, but are not limited to, respiratory distress, septicshock, cardiogenic shock, hypovolemic shock, atherosclerosis,hyperhomocysteinemia, venous thrombosis, arterial thrombosis, coronaryocclusion, pulmonary embolism, cerebrovascular accidents, vascularfibrosis, ectopia lentis, osteoporosis, mental retardation, skeletaldeformities, pulmonary hypertension, malignancy, infections,inflammation, asthma, tolerance to narcotics and central nervous systemdisorders. Furthermore, the use of these methods is not limited to thesediseases. This method can be of use in assaying biologically activenitric oxide equivalents in any disease state or medical disorder inwhich nitric oxide is implicated.

The data set forth in the Examples below demonstrate that adetermination of NO bound to hemoglobin as nitrosylhemoglobin and SNO-Hbcan be used to assess the efficiency of oxygen delivery to the tissuesof an animal or a human patient. Values for nitrosylhemoglobin andSNO-Hb in blood can be determined together, in one method, or they canbe determined in separate methods. An additional determination foroxygen in the blood, as measured by methods known in the art, can beused in conjunction with determinations of nitrosylhemoglobin and SNO-Hbconcentrations, to assess oxygen delivery to a site in the body fromwhich a blood sample is taken.

Further Embodiments

The subject invention relates to a method of loading cells with anitrosating agent as exemplified for red blood cells as in FIG. 3A, butwhich can be accomplished in more ways. Suitable conditions for pH andfor the temperature of incubation are, for example, a range of pH 7-9,with pH 8 being preferred, and a temperature range of 25 to 37° C. Forred blood cells, short incubation times of 1 to 3 minutes are preferredfor limiting the formation of S-nitrosylated forms of Hb. However,intracellular concentrations of 1 mM nitrosating agent can be reached.

The nitrosating agent should be a good donor of NO⁺ and should be ableto diffuse through the cell membrane of the target cell type. That is,it is preferably of low molecular weight, compared to the molecularweight of S-nitrosoproteins. Examples are S-nitroso-N-acetylcysteine,S-nitrosocysteinylglycine, S-nitrosocysteine, S-nitrosohomocysteine,organic nitrates and nitrites, metal nitrosyl complexes, S-nitro andS-nitroso compounds, thionitrites, diazeniumdiolates, and other relatednitrosating agents as defined in Feelisch, M. and Stamler, J. S.,“Donors of Nitrogen Oxides” chapter 7, pp. 71-115 In Methods in NitricOxide Research (Freelisch, M. and Stamler, J. S., eds.) John Wiley andSons, Ltd., Chichester, U. K. (1996), the contents of which chapter arehereby incorporated by reference in their entirety. Nitrosating agentshave differential activities for different reactive groups onmetal-containing proteins. A nitrosating agent can be chosen for minimaloxidation of the heme iron of Hb, and maximum activity in nitosylatingthiol groups such as found on cysteine. Assay methods are available fordetection of nitrosation products, including S-nitrosothiols. SeeStamler et al., U.S. Pat. No. 5,459,076, the contents of which arehereby incorporated by reference in their entirety. See also, forexample, Keefer, L. K., and Williams, D. L. H., “Detection of NitricOxide Via its Derived Nitrosation Products,” chapter 35, pp. 509-519 InMethods in Nitric Oxide Research (Freelisch, M. and Stamler, J. S.,eds.) John Wiley and Sons, Ltd., Chichester, U.K., 1996; see alsoStamler, J. S. and Feelisch, M., “Preparation and Detection ofS-Nitrosothiols,” chapter 36, pp. 521-539, ibid. Nitrite and nitrateproducts can be assayed by methods described, for instance, in Schmidt,H.H.H.W. and Kelm, M., “Determination of Nitrite and Nitrate by theGriess Reaction,” chapter 33, pp. 491-497, ibid., and in Leone, A. M.and Kelm, M., “Capillary Electrophoretic and Liquid ChromatographicAnalysis of Nitrite and Nitrate,” chapter 34, pp. 499-507, ibid.

Such low molecular weight nitrosating agents can be used in red bloodcells to deliver NO-related activity to tissues. Treatment of red bloodcells with nitrosating agent further serves to increase the O₂ deliverycapacity of red blood cells. Such treatment of red blood cells alsoallows for the scavenging of free radicals, such as oxygen freeradicals, throughout the circulation. It is possible to load red bloodcells with S-nitrosothiol, for example, by a process of removing wholeblood from a patient's body (as a minimal method of isolating red bloodcells), treating the red blood cells with low molecular weightnitrosating agent, such as by incubating the red blood cells in asolution of S-nitrosothiol, and then reintroducing the red blood cellsinto the same patient, thereby allowing the treatment of a number oftypes of diseases and medical disorders, such as those which arecharacterized by abnormal O₂ metabolism of tissues, oxygen-relatedtoxicity, abnormal vascular tone, abnormal red blood cell adhesion,and/or abnormal O₂ delivery by red blood cells. Such diseases caninclude, but are not limited to, ischemic injury, hypertension, shock,angina, stroke, reperfusion injury, acute lung injury, sickle cellanemia, and blood borne infectious diseases such as schistosomiasis andmalaria. The use of such “loaded” red blood cells also extends to bloodsubstitute therapy and to the preservation of living organs, such asorgans for transplantation. In some cases, it will be appropriate totreat a patient with loaded red blood cells originating from a differentperson.

A particular illustration of the mechanism of the treatment method ispresented here by considering sickle cell anemia. Sickle cell patientssuffer from frequent vascular occlusive crises which manifest inclinical syndromes such as the acute chest syndrome and hepaticdysfunction. Both endothelial cell dysfunction, resulting in a clottingdiathesis as well as dysfunction intrinsic to the red blood cell, arecentral to disease pathogenesis. At the molecular level, the increasedexpression of vascular adhesion molecules such as VCAM promote theadhesion of sickled red blood cells containing abnormal hemoglobin. Itfollows that decreasing cytokine expression on endothelial cells,promoting endothelial function and attenuating red cell sickling, arekey therapeutic objectives. However, currently used therapies have beengenerally unsuccessful.

In this novel method for loading red blood cells with intracellularNO-donor S-nitrosothiols, the effect is to increase oxygenaffinity—which in and of itself should attenuate red blood cellsickling—and to endow the red blood cell with vasodilator andantiplatelet activity, which should reverse the vasoocclusive crisis.Moreover, nitric oxide should attenuate the expression of adhesionmolecules on endothelial cell surfaces, thus restoring endothelialfunction.

Herein is described a novel therapeutic approach to the treatment ofsickle cell disease which involves loading of red blood cells withS-nitrosothiols or other nitrosating agents. Two examples of therapeuticapproaches are given. In the first, the patient's own red blood cellsare S-nitrosated extracorporeally (yielding “loaded” red blood cells)and then given to the patient. The second approach is to directlyadminister to a patient an agent such as S-nitrosocysteine, which ispermeable to red blood cells.

For some diseases or disorders, the administration of NO-loaded redblood cells is especially desirable. Upon a change from the oxygenatedto the deoxygenated state, or upon a change in the oxidation state ofthe heme Fe from the reduced state (FeII) to the oxidized (FeIII) state,NO is released from the thiol groups of hemoglobin, and is rapidlytransferred to glutathione to form S-nitrosoglutathione. Red blood cellsare known to have a high concentration of glutathione.S-nitrosoglutathione efficiently delivers NO to tissues.

In another aspect, the invention is a method for the treatment ofinfection by administering to an infected mammal an agent which causesS-nitrosation of thiol groups within the cells which are the target ofsuch agent. For example, an S-nitrosothiol to which lymphocytes arehighly permeable can be administered to a patient infected with HIV.Such treatment for HIV can also be used extracorporeally, to bloodisolated from the patient. In another application, the infection isbacterial, and the S-nitrosothiol to be used as an anti-bacterial agentis one to which the target bacterial cells are highly permeable, ascompared to the permeability properties of the host cells. (See, forexample De Groote, M. A., et al., Proc. Natl. Acad. Sci. USA92:6399-6403 (1995).) Alternatively, nitrosothiols can be used to treatPlasmodium falciparum within red blood cells.

Another embodiment of the invention is a method for specificallymodifying a protein containing one or more metal atoms so that theprotein becomes S-nitrosylated at one or more thiol groups withoutmodifying the metal, as by changing the oxidation state or causing themetal atoms to bind NO. This can be accomplished by the use of a reagentwhich possesses NO⁺ character, such as a nitrosothiol (See, forinstance, Example 4A.), which reacts specifically with thiol groups of aprotein in which metal is bound.

An S-nitrosation method has been devised which does not affect the hemeof hemoglobin. SNO-Hb (SNO-Hb(FeII)O₂) can be synthesized fromHb(FeII)O₂ with up to 2 SNO groups per tetramer without oxidation of theheme Fe from FeII to FeIII. In contrast, when Hb(FeII)O₂ is incubatedwith excess nitric oxide or nitrite, methemoglobin (HbFe[III]) formsrapidly (Example 1B) and to a significant extent. When Hb[FeII] isincubated with nitric oxide, NO binds rapidly to the heme, formingHb(FeII)NO to a significant extent (Example 1A).

Although rates of formation of SNO-Hb(FeII)O₂ from Hb(FeII) O₂ are morerapid (see Example 2A), the corresponding SNO-deoxyHb form can also bemade by incubation of S-nitrosoglutathione or S-nitrosocysteine, forexample, with Hb(FeII), yielding SNO-Hb(FeII), as in Example 1C.

The effects of the various forms of Hb on vasodilation—constriction,dilation or a neutral effect—depend on three factors: whether 1) the Feof the heme is oxidized, 2) O₂ is bound at the heme (that is, theoxygenation state, dictated by the conformation of the protein as Rstate or T state), and 3) thiol is present in sufficient concentrationto facilitate the transfer of NO⁺.

The importance of the first factor is shown in FIG. 4A. Hb(FeII)O₂ andSNO-Hb[FeII]O₂ act as vasoconstrictors, but SNO-Hb[FeIII] (metHb form,where FeII has been oxidized to FeIII) acts as a vasodilator. FIG. 4Ashows that SNO-Hb[FeII]O₂ with oxygen bound at the heme, and with aratio of SNO/Hb=2, acts as a powerful vasoconstrictor.

SNO-Hb(FeII) is also a vasodilator. FIG. 2B illustrates the secondfactor in demonstrating that rates of RSNO decomposition and transferare much faster for SNO-Hb in the deoxy state than for SNO-Hb in the oxystate.

It can be seen how the NO⁺-donating properties of SNO-Hb depend onoxygen concentrations. SNO-Hb releases oxygen at sites of low oxygenconcentration or under oxidizing conditions. SNO-Hb releases its NOgroup(s) to cause vasodilation either due to 1) oxidation of the heme Feto FeIII or 2) loss of the O₂ from the heme by deoxygenation. It isshown in FIG. 2B that NO is transferred off SNO-Hb best in the deoxystate. In ischemia, SNO-Hb deoxygenates, rapidly followed by the loss ofNO. It can be seen from the data that SNO-metHb having a ratio of 1SNO/SNO-metHb is a more powerful vasodilator than SNO-oxyHb having aratio of 2 SNO/SNO-oxyHb. It should be noted that S-nitrosation of Hbinduces the R state (oxy conformation). Thus, it follows that 1SNO-oxyHb molecule having a ratio of 1 SNO/SNO-oxyHb is less potent than10 molecules of SNO-oxyHb having a ratio of 0.1 SNO/SNO-oxyHb.

The third factor is illustrated by the results shown in FIG. 4B. Theseresults demonstrate potentiation by thiol of the vasodilator effect ofSNO-Hb(FeII)O₂ and SNO-Hb(FeIII). Transfer of NO⁺ from SNO-Hb to lowmolecular weight nitrosothiols is more efficient when Hb is in the deoxystate compared to the oxy state (FIG. 2B) or in the met state comparedto the oxy state (FIG. 4C).

NO is released or transferred as NO⁺ (nitrosyl cation) from SNO-Hb. TheSNO groups of SNO-Hb have NO⁺ character. Transfer of NO⁺ from SNO-Hboccurs most efficiently to low molecular weight thiols, such asglutathione, and is most efficient when the heme is oxidized (SNO-metHb)or the SNO-Hb is in the deoxy state.

One embodiment of the invention resulting from these findings is amethod of therapy that enhances the transfer of NO⁺ from SNO-Hb to lowmolecular weight thiols, thereby delivering NO biological activity totissues, by the coadminstration of low molecular weight thiols, alongwith a form of SNO-Hb, to a mammal in need of the physiological effectsof NO. To further increase the effect of NO release it is preferred thatthe SNO-forms of metHb or deoxyHb (or an equivalent conformation or spinstate) be administered with the thiol (See FIG. 2B, for example.) Amixture of SNO-metHb and SNO-oxyHb, and possibly also thiol, can also beused. The composition and proportion of these components depends on thedisease state. For example, to achieve both enhanced O₂ delivery and NOdelivery, SNO-oxyHb can be used. Where no further delivery of O₂ isdesirable, as in acute respiratory distress syndrome, for example, theSNO-forms of metHb and deoxyHb are especially preferred. Alternatively,the ratios of SNO/Hb can be regulated to control O₂ release.

A further invention arising out of the discoveries presented herein is amethod for preserving a living organ ex vivo, for example fortransplantation, comprising perfusing the organ with a compositioncomprising nitrosated hemoglobin and low molecular weight thiol or NOdonating agent, wherein SNO-Hb(FeII)O₂ is a preferred nitrosatedhemoglobin.

The vessel ring bioassay data of FIG. 4A agree well with the in vivodata of FIG. 5. The results of the experiments described in Example 5confirm that Hb(FeII)O₂ (oxyHb) causes an increase in blood pressure invivo, as it did also in vitro. SNO-Hb(FeIII) (SNO-metHb) causes adecrease in blood pressure in vivo as well as in vitro. SNO-Hb(FeII)O₂(SNO-oxyHb) has a negligible effect on blood pressure in vivo incontrast to the increase in tension seen in the corresponding vesselring bioassay. For SNO-oxyHb the in vivo effect is neutral. This isexplained by the constrictive effect caused by NO becoming bound to theheme being compensated by the release of NO upon deoxygenation.Therefore, SNO-oxyHb can deliver O₂ with minimal effect on bloodpressure.

With knowledge of the results herein it is possible to synthesize Hbproteins with predicted NO-releasing properties, which will constrict,dilate, or have no effect on blood vessels. An additional option is thechoice between making oxygenated or deoxygenated forms to administer formedical conditions in which O₂ delivery is desirable, or undesirable,respectively.

It is possible to produce a variety of modified Hbs having specificdesired properties of O₂ and NO delivery. For example, Hb in the R stateor R-structure (oxyHb) can be converted to the T state or T-structure(deoxyHb) by a number of known methods. This can be done, for example,by reaction of Hb with inositol hexaphosphate. It is also known to thoseskilled in the art that Hb in the R state can be made, for example, bytreating Hb with carboxypeptidase. Similarly, it is known that metHb canbe synthesized using ferricyanide or nitrite.

Producing Hb molecules which are locked in the T state allows thesynthesis of RSNO-Hb which remains in a form that is a biologicallyactive donor of NO, rather than a carrier of NO. Hb which is locked inthe R state can be used as a substrate for the synthesis of RSNO-Hbwhich carries a maximum amount of NO per molecule.

Another embodiment of the invention is a blood substitute comprising oneor more forms of Hb which have been specifically S-nitrosated to someextent at one or more thiol groups of the Hb, in order to regulate O₂release and NO release. Conditions to be treated include those in whichNO or O₂ delivery is desired, those in which NO or O₂ utilization isdesired, or those in which NO or O₂ is in excess. For example, in amedical condition which is characterized by the presence of an excess ofoxygen free radicals and excess NO⁺, both the heme of SNO-Hb and NOreleased by SNO-Hb serve to trap oxygen free radicals. The heme Fe isoxidized in the process of scavenging oxygen free radicals and NO⁺, andNO is released from the oxidized Hb by donation to a thiol, in the formof RSNO⁺, which is not toxic. Inflammation and reperfusion injury, forexample, are characterized by excess NO production and an excess ofoxygen free radicals. Forms of Hb scavenge oxygen radicals and free NO,converting NO to forms that are not toxic.

A further embodiment of the invention is a method of therapy for acondition that would benefit from the delivery of NO in a biologicallyactive form or O₂ or both, based on the administration of a bloodsubstitute comprising a form of nitrosated Hb, such asS-nitrosohemoglobin, either alone or in combination with a low molecularweight thiol, for example. For example, SNO-Hb is useful to treatmyocardial infarction. SNO-Hb has the effect of donating NO, keepingblood vessels open. SNO-Hb deoxygenates at low oxygen tension,delivering oxygen and releasing NO at the same site, thereby causingvasodilation. (See Example 7 and FIGS. 6A-6F.) These effects can beaugmented by also administering thiol, either simultaneously withSNO-Hb, or before or after. For the purpose of treating myocardialinfarction, for example, a high concentration or dose of SNO-Hb that hasa low ratio of SNO/SNO-Hb is appropriate. Alternatively, SNO-metHb canbe used for this purpose. A further application of these principals is amethod for increasing cerebral blood flow in a mammal comprisingadministrating to the mammal a composition comprisingS-nitrosohemoglobin, as illustrated in FIGS. 23A-23I.

In another aspect, the invention is a method of enhancing NO-donortherapy by coadministering a composition comprising SNO-Hb or otherforms of nitrosated Hb together with a nitroso-vasodilator(nitroglycerin, for example) which would be otherwise consumed by theconversion of oxyHb to metHb in Hb which has not been S-nitrosated. Acomposition comprising a low molecular weight thiol can have the effectof producing a vasorelaxant response in a mammal (see Example 22 andFIG. 20D).

Platelet activation is manifested by a number of events and reactionswhich occur in response to adhesion of platelets to a nonplateletsurface such as subendothelium. Binding of agonists such as thrombin,epinephrine, or collagen sets in motion a chain of events whichhydrolyzes membrane phospholipids, inhibits adenylate cyclase, mobilizesintracellular calcium, and phosphorylates critical intracellularproteins. Following activation, platelets secrete their granule contentsinto plasma, which then allow the linking of adjacent platelets into ahemostatic plug. (See pages 348-351 in Harrison's Principles of InternalMedicine, 12th edition, eds. J. D. Wilson et al., McGraw-Hill, Inc., NewYork, 1991).

A thrombus is a pathological clot of blood formed within the circulatorysystem. It can remain attached to its place of origin or becomedislodged and move to a new site within the circulatory system.Thromboembolism occurs when a dislodged thrombus or part of a thrombuspartially or completely occludes a blood vessel and prevents oxygentransport to the affected tissues, ultimately resulting in tissuenecrosis.

Sites where damage has occurred to the vascular surface are especiallysusceptible to the formation of thrombi. These sites include those onthe interior surface of a blood vessel in which damage to theendothelium, narrowing or stenosis of the vessel, or atheroscleroticplaque accumulation has occurred.

NO is one of several endothelium-derived thromboregulators, which aredefined as physiological substances that modulate the early phases ofthrombus formation. In particular, No reduces platelet adhesion,activation and recruitment on the endothelial cell surface, and achievesthis, it is thought, by activating platelet guanylate cyclase, therebyincreasing platelet intracellular cGMP (Stamler, J. S. et al, Circ. Res.65:789-795 (1989)), and decreasing intraplatelet Ca²⁺ levels. NO and theprostacylcin prostaglandin (PG) I₂ act synergistically to inhibit andactively mediate platelet disaggregation from the collagen fibers of thesubendothelial matrix. Unlike prostacyclin, NO also inhibits plateletadhesion. Furthermore, platelets synthesize NO, and the L-arginine-NOpathway acts as an intrinsic negative feedback mechanism to regulateplatelet reactivity. NO is involved in leukocyte interactions with thevessel wall and can inhibit neutrophil aggregation. (See review article,Davies, M. G. et al., British Journal of Surgery 82:1598-1610, 1995.)

NO is antiathrogenic in a number of ways. (See, for example, Candipan,R. C. et al., Arterioscler. Thromb. Vasc. Biol. 16:44-50, 1996.) NOinhibits smooth muscle proliferation and attenuates LDL (low densitylipoprotein) oxidation and other oxidant-related processes.

Hemoglobin may promote atherosclerosis as well as thrombosis as aconsequence of its No-scavenging property. This limitation of hemoglobinderives from its high affinity for nitric oxide. In vitro, NO is apotent inhibitor of platelet aggregation and adhesion to collagenfibrils, the endothelial cell matrix and monolayers (Radomski, M. W. etal., Br. J. Pharmacol. 92:181-187 (1987); Radomski, M. W. et al., Lancet2:1057-1058 (1987); Radomski M. W. et al., Biochem. Biophys. Res.Commun. 148:1482-1489 (1987)). NO elevates cGMP levels in platelets,thereby decreasing the number of platelet-bound fibrinogen molecules andinhibiting intracellularCa^(++ flux and platelet secretion (Mellion, B. T. et al.,) Blood57:946-955 (1981); Mendelson, M. E. et al., J. Biol. Chem.165:19028-19034 (1990); Lieberman, E. et al., Circ. Res. 68:1722-1728(1991)). Scavenging of nitric oxide by Hb prevents the molecule frominhibiting platelets. This explanation has been given support by in vivostudies (Krejcy, K. et al., Arterioscler. Thromb. Vasc. Biol.15:2063-2067 (1995)).

The results shown in FIGS. 7A-7C (see Example 9) show that nitrosatedhemoglobins, including SNO-Hb, can be used in the treatment of acuteblood clotting events that occur as a result of increased plateletdeposition, activation and thrombus formation or consumption ofplatelets and coagulation proteins. Such complications are known tothose of skill in the art, and include, but are not limited tomyocardial infarction, pulmonary thromboembolism, cerebralthromboembolism, thrombophlebitis and unstable angina, and anyadditional complication which occurs either directly or indirectly as aresult of the foregoing disorders.

SNO-Hb and other nitrosated hemoglobins can also be usedprophylactically, for example, to prevent the incidence of thrombi inpatients at risk for recurrent thrombosis, such as those patients with apersonal history or family history of thrombosis, with atheroscleroticvascular disease, with chronic congestive heart failure, withmalignancy, or patients who are pregnant or who are immobilizedfollowing surgery.

NO is known to activate soluble guanylate cyclase, which produces cGMP.cGMP mediates inhibition of platelet aggregation. Results in Example 10demonstrate that this inhibition of platelet aggregation may be mediatednot by cGMP alone, but by some other mechanism as well.

Certain compounds or conditions are known to cause a shift in theallosteric equilibrium transition of Hb towards either of the twoalternative quaternary structures of the tetramer, the T- orR-structures. (See, for example, pages 7-28 in Perutz, M., Mechanisms ofCooperativity and Allosteric Regulation in Proteins, CambridgeUniversity Press, Cambridge, U.K., 1990.) These are, for instance, theheterotropic ligands H⁺, CO₂, 2,3-diphosphoglycerate (2,3-DPG) and Cl⁻,the concentrations of which modulate oxygen affinity. The heterotropicligands lower the oxygen affinity by forming additional hydrogen bondsthat specifically stabilize and constrain the T-structure. Othercompounds affecting the allosteric equilibrium include inositolhexaphosphate (IHP) and the fibric acid derivatives such as bezafibrateand clofibrate. The fibric acid derivatives, antilipidemic drugs, havebeen found to combine with deoxy-, but not with oxyhemoglobin. Theystabilize the T-structure by combining with sites in the central cavitythat are different from the DPG binding sites. Other allostericeffectors have been synthesized which are related to bezafibrate. Aligand that stabilizes specifically the R-structure increases the oxygenaffinity, and a ligand that stabilizes the T-structure does the reverse.Other ligands can affect the spin state of the heme. For example, indeoxyhemoglobin and in methemoglobin the Fe is high-spin ferrous (S=2)and 5-coordinated; in oxyhemoglobin and in cyan-metHb the Fe is low-spinferrous (S=0) and 6-coordinated; when H₂O is the sixth ligand,methemoglobin is also high-spin. The inhibition of platelet aggregationby S-nitroso-methemoglobin seen in FIG. 7C is consistent with enhancedpotency in the high spin conformation. Substances which control theallosteric equilibrium or spin state of hemoglobin can be administeredin a pharmaceutical composition to a human or other mammal, in atherapeutically effective amount, to promote the formation of, or tostabilize, a particular allosteric structure and/or spin state ofhemoglobin, thereby regulating platelet activation, e.g., by convertinghemoglobin from R-structure to T-structure.

The dosage of Hb required to deliver NO for the purpose of plateletinhibition can be titrated to provide effective amounts of NO withoutcausing drastic changes in blood pressure. If the goal of the therapy isto deliver oxygen, the Hb can be administered in a unit of blood toavoid a drop in blood pressure. If the goal is to alleviate shock, verylittle Hb can be administered compared to the amount to be given formyocardial infarction. For shock, the more important goal is to deliverNO rather than to deliver oxygen. For this objective, it can bepreferable to use continuous infusion or several infusions per day.Example 12 (see FIG. 10) shows that the effects of SNO-Hb(FeII)O₂ onblood flow in rat brain last over 20 minutes; in other experiments aneffect has been seen for up to an hour. There is a correlation betweenblood pressure effects and platelet inhibition effects, but plateletinhibition occurs at a lower NO concentration than that which isrequired to produce blood pressure effects, and generally lasts longer.

Example 11 shows that S-nitrosothiols can be used to add NO groups notonly on the thiol groups of cysteine residues in hemoglobin, but also onother reactive sites of the hemoglobin molecule. The products of thenitrosation reactions in Example 11 were hemoglobin molecules with morethan 2 NO groups per Hb tetramer. The exact sites of the addition of NOhave not been confirmed, but it is expected that NO addition occurs atthiol groups and various other nucleophilic sites within Hb, includingmetals. Reactive sites, after the thiol groups, are tyrosine residuesand amines, and other nucleophilic centers.

Nitrosation reactions on other proteins have been investigatedpreviously (Simon, D. I. et al., Proc. Natl. Acad. Sci. USA 93:4736-4741(1996)). Methods of modifying proteins to produce nitrosoproteins areknown in the art, and include, for example, exposing the protein toNaNO₂ in 0.5 M HCl (acidified NO₂ ^(−) for) 15 minutes at 37° C. Analternative method is to place a helium-deoxygenated solution of proteinin 100 mM sodium phosphate, pH 7.4, inside dialysis tubing and exposethe protein to NO gas bubbled into the dialysate for 15 minutes.(Stamler, J. S. et al., Proc. Natl. Acad. Sci. USA 89:444-448 (1992);see also Williams, D. L. H. Nitrosation, Cambridge University Press, NewYork (1988), which gives further methods of nitrosation).

By these methods, multiple NO-related modifications (“NO groups” or “NObiological equivalents” resulting from nitrosations, nitrosylations ornitrations) can be made on Hb at nucleophilic sites, which can includethiols, nucleophilic oxygen atoms as found in alcohols, nucleophilicnitrogen atoms as found in amines, or the heme iron. Agents facilitatingnitrosations, nitrosylations or nitrations of Hb can be thought of as“NO or NO⁺ donating agents.” The products of such modifications may havesuch groups, for example, as —SNO, —SNO₂, —ONO, ONO₂, —CNO, —CNO₂, —NNO,—NNO₂, —FeNO, —CuNO, —SCuNO, SFeNO and the different ionized forms andoxidation variants thereof. (See, regarding oxidation of hemoglobin byCu⁺⁺, Winterbourne, C., Biochemistry J. 165:141-148 (1977)). Thecovalent attachment of the NO group to sulfydryl residues in proteins isdefined as S-nitrosation; the covalent attachment of the NO group to ametal, such as Fe, can be called nitrosylation, yielding ametal-nitrosyl complex. General NO attachment to nucleophilic centers isreferred to herein as nitrosation. Thus, the term nitrosated hemoglobinas used herein includes SNO-Hb and Hb(FeII)NO as well as other forms ofhemoglobin nitrosated at other sites in addition to thiols and metals.In addition, Hb can be nitrated. Hbs which have been nitrosated and/ornitrated at multiple different types of nucleophilic sites (termedpolynitrosated, that is, having NO equivalents added to othernucleophilic sites as well as to thiols; or polynitrated, respectively)will permit transnitrosation reactions and the release of NO and itsbiological equivalents in the circulatory system at different rates andengendering different potencies.

These and other nitrosation and nitration reactions can cause oxidationof the heme Fe to some extent, under some conditions. However, someminor degree of oxidation is acceptable. The nitrosated Hb is still beuseful as a therapeutic agent if oxidized to a minor extent. Forapplications where the NO-delivering function, rather than theO₂-delivering function of nitrosated Hb, is more desirable, extensiveoxidation of the heme Fe is acceptable.

If it is desired to avoid oxidation of the heme Fe, it is possible toremove the heme, perform the necessary chemical reactions upon theprotein to nitrosate to the extent desired, and replace the heme intothe modified hemoglobin product. (See, for removing and replacing theheme, Antonini, E. and Brunori, M., Hemoglobin and Myoglobin in theirReactions with Ligands, Elsevier, New York, 1971.)

In addition to the nitrosating under conditions that do not oxidize theheme, such as brief exposure to low molecular weight RSNOs, asillustrated in Examples 1 and 2, alternative methods can be used toproduce nitrosated hemoglobin in which the heme Fe is not oxidized. Forinstance, it is possible to produce by recombinant methods α and βglobin chains, nitrosate them to the extent desired, then assemble thechains with heme to form a functional, nitrosated tetramer. (See, forexample, European Patent Application EPO 700997, published Mar. 13,1996, “Production in bacteria and yeast of hemoglobin and analoguesthereof.”)

Another alternative method to nitrosate the α and β globin chainswithout producing a form of methb as the end product, is to nitrosatethe intact Hb molecule to the extent desired, thereby allowing the hemeFe to be oxidized, then reduce the heme Fe from FeIII to FeII bytreating the nitrosated Hb with either methemoglobin reductase or acyanoborohydride such as sodium cyanoborhydride.

It has been generally thought that nitric oxide as NO gas in solutionreacts with hemoglobin (Hb) in two major ways: 1) with the deoxyHb toform a stable nitrosyl (FeII) heme adduct; and 2) with oxyHb to formnitrate and metHb—a reaction that inactivates NO. These two reactionscontributed to the idea that Hb is a scavenger of NO. In both of thesereactions, NO biological activity is lost. The results described hereindemonstrate that, in fact, neither reaction occurs under physiologicalconditions. Rather, the products of the NO/Hb reaction are dictated bythe ratio of NO to Hb, and by the conformation of Hb—R(oxy) vs.T(deoxy).

At low ratios of NO to deoxyHb (e.g., 1:100 or less), the Hb molecule isin T-structure. Under this condition, NO introduced as gas to a Hbsolution binds to the α-hemes, as has been seen by EPR. Uponintroduction of oxygen, with conversion to the R state, NO istransferred to a thiol of cysteine to yield S-nitrosohemoglobin withclose to 100% efficiency. At ratios of NO/Hb of 1:25-1:50, theefficiency of formation of SNO-Hb is ˜35% (decreasing with increasingNO/Hb ratio). The reaction appears to involve migration of NO from aheme to β heme and then to the β thiol. In going from the heme to athiol, the heme or nitrosothiol needs to lose an electron by oxidation(NO→NO⁺ or RSNO.→RSNO). Oxygen serves as an electron acceptor in thesystem, driving the reaction thermodynamically, as well as causing aconformational change by its binding at the heme, which exposes thethiol groups. At higher ratios of NO to Hb (1:20-1:2), with the proteinstill in T-structure, the protein liberates NO⁻ from the hemes withproduction of metHb. This occurs in the absence of O₂ and providesanother indication that the NO bound to β-hemes is unstable. Once O₂ isintroduced, S-nitrosothiol (SNO) forms, but the relative yield is verylow because of loss to NO⁻. The yield of SNO-Hb approaches zero at NO/Hbratios of 1:2, upon introduction of oxygen.

At the higher ratios of NO to Hb (i.e., >0.75-1), NO itself maintainsthe R-structure. Under this condition, the NO is more stable because ofan unusual constraint on the molecule. Specifically, loss of NO from theβ hemes promotes the T-structure, whereas formation of SNO-Hb selectsfor the R-structure. This is not a favored reaction. The consequence isthat small amounts of S-nitrosohemoglobin are formed, but the yields arelow (−5%). This does not exclude the possibility that the molecule hastherapeutic value.

The reaction of NO with oxyHb is also dependent on the ratios of NO tooxyHb. Under conditions of relatively high (non-physiological) ratios ofNO to Hb, (NO/oxyHb>1:20), NO appears to destabilize the hydrogen bondbetween the O₂ and the proximal histidine (by competing for it) yieldingsome metHb. By changing the ionic composition of the solvent buffer(e.g., borate 0.2 M, pH 7.4), formation of metHb can be significantlyreduced even with excess NO (NO/Hb=3:1). On the other hand, metHbformation is facilitated in acetate buffer at pH 7.4; when the hydrogenbond between O₂ and the proximal histidine is broken, the O₂ seems togain superoxide-like character. NO then reacts rapidly to form metHb andnitrate. Efficient methb formation actually requires an excess ofNO/oxyHb. In contrast, at lower ratios of NO/Hb (<1:20), it reacts withthe small residual fraction (<1%) of deoxyHb, in turn producingS-nitroso-hemoglobin extremely efficiently. As the concentration of NOis increased, there is some reaction with oxyHb, but the products arenitrite and nitrate, not nitrate alone. The conclusion is that NO can beincubated in reaction mixtures of oxyHb without inactivating the O₂binding functionality by converting it to nitrate.

Nitrosylhemoglobin can be used in an animal or human as a therapeutic NOdonor for the prevention or treatment of diseases or medical disorderswhich can be alleviated by delivery of No or its biologically activeform to tissues affected by the disease or medical disorder. LikeSNO-Hb, nitrosylhemoglobin can be administered as a blood substitute,because nitrosylhemoglobin can be converted to SNO-Hb underphysiological conditions. No is released from the thiol either bydeoxygenation or by conversion to metHb.

Inhaled NO causes selective pulmonary vasodilation without influencingsystemic responses. A previously-formed rationale behind its use is thatscavenging by Hb prevents adverse systemic effects. It is illustrated inExamples 14-21 that NO can be used to produce S-nitrosohemoglobin, whichis a potent vasodilator and antiplatelet agent. Inhaled NO can be usedto raise levels of endogenous S-nitrosohemoglobin. Similarly, treatmentof red blood cells (RBCs) with NO can be used to form SNO-RBCs, or“loaded” red blood cells.

Compared to SNO-deoxyHb, which is a good No donor, but which wouldrelease its No very quickly, or SNO-oxyHb, which would release its NOmore slowly, but has a propensity to form metHb over time,nitrosyl-deoxyhemoglobin stored in a form such that final ratio ofNO:heme is less than about 1:100 or greater than about 0.75, is stable.Formation of methb is prevented at these NO:heme ratios. For this reasonnitrosyl-deoxyhemoglobin stored with such NO:heme ratios in aphysiologically compatible buffer can be administered to an animal orhuman as an NO donor. Erythrocytes comprising nitrosylhemoglobin canalso be used as NO donors. Erythrocytes comprising nitrosylhemoglobincan be made in a process comprising incubating deoxygenated erythrocytesin a solution comprising No.

A blood substitute or therapeutic which can be used as an NO donor, andwhich is free of the vasoconstrictor effects of underivatized Hbs, canbe made by obtaining a solution of oxyHb (including solutions stored inthe form of oxyHb) and adding NO as dissolved gas, yielding SNO-oxyHb.Buffer conditions and NO:Hb ratios can be optimized, as illustrated inExample 21 and FIG. 19, to yield S-nitrosothiol without significantproduction of oxidized Hb (metHb). For example, NO added tooxyhemoglobin in 10 mM phosphate buffer, pH 7.4, at a ratio of less than1:30 NO:Hb resulted in formation of SNO-oxyHb with minimal formation ofmetHb. This ratio can be increased by varying the buffer conditions, forexample by the use of 10 mM phosphate, 200 mM borate at pH 7.4. Thebuffer anions as well as the buffer concentration should be chosencarefully. For instance, acetate and chloride have the opposite effectfrom borate, increasing the formation of metHb and nitrite at 200 mM, pH7.4.

This can be explained by a competition between free NO and oxygen for aH-bond with the imidazole of the proximal His residue. If lowconcentrations of NO are used, in low ionic strength buffer, e.g., 10 mMphosphate, metHb does not readily form. If the H-bond is weakened byincreasing the ionic strength of the buffer, NO reacts more readily withoxyHb, yielding more metHb. Buffers with a low pK_(a) relative to pH 7.4tend to stabilize FeIII. Buffers having a pK_(a) at least about two pHunits higher than the reaction condition are preferred.

A blood substitute can be made which acts as a donor of NO⁻. NO can beadded to a solution of deoxyHb at a ratio of NO:Hb in the range of 1:100to 1:2, with a ratio of NO/heme of approximately 1:10 being preferred.If the ratio of NO:heme is increased, to a NO:Hb of about 2 (at which Hbis still in the T (deoxy) state), in the absence of an electronacceptor/free radical scavenger, NO is released from the β heme as NO⁻,with oxidation of the heme iron to form metHb. The product solution canbe used as a blood substitute or a therapeutic NO donor. NO⁻ can protectfrom N-methyl-D-glutamate-mediated brain injury in stroke; this effecthas not been found for NO.

Nitosylhemoglobin belongs to a broader class of nitrosyl-heme-containingdonors of NO which can be administered to an animal or human for thedelivery of NO to tissues. Nitrosyl-heme-containing donors of NOinclude, for example, the nitrosated (“nitrosated” as defined herein)hemoglobins nitrosylhemoglobin and SNO-nitrosylhemogobin, nitrosyl-heme,and substituted forms of hemoglobin in which a different metal, (e.g.,Co⁺⁺, Mg⁺⁺, Cu⁺⁺) is substituted for the heme iron, ornitrosyl-porphyrins are substituted for the heme.

Applicants teach physiologically significant results that provide arationale for NO donors to be attached to Hb. Such derivatized Hbs canthemselves serve as NO-donating therapeutics and can ameliorate the sideeffects of underivatized Hb administered as a blood substitute, forexample. At one time, it had been thought that there would be no use forthese compounds, because it was thought that NO released by the Hb wouldimmediately be scavenged by the heme. It had been thought also that thereleased NO would oxidize Hb and limit oxygen delivery. The samerationale has previously limited the administration of NO donors, suchas nitroglycerin and nitroprusside, because they had been thought tocause the formation of metHb.

Preferably, NO-donors to be covalently attached to hemoglobin arerelatively long-lived and have at least one functional group that can beused for the chemical attachment to hemoglobin. Examples of NO-donorsinclude nitroprusside, nitroglycerin, nitrosothiols, and thediazeniumdiolte class of compounds (also called “NONOates”) havingstructure 1.

A variety of these compounds have been synthesized that, in theiranionic form, release NO without activation at physiological pH (Keefer,L. K. et al., Am. Chem. Soc. Symposium Ser. 553:136-146 (1994); Hanson,S. R. et al., Adv. Pharmacol. 34:383-398 (1995)). Systemicadministration can result in system-wide effects, according toequation 1. However, attachment to hemoglobin can be used to producetissue-selective delivery of NO and oxygen. For instance, covalentlyesterifed NO-donors can be activated predominantly in the liver.Different NO donors can be chosen to be linked to hemoglobin fordifferent controlled release rates of NO from Hb.

Compound 4 (below), for example, is a diazeniumdiolate with a half-lifefor NO release, at 37° C. and pH 7.4, of approximately two weeks. It canbe converted to its nucleophilic N-4 mercaptoethyl derivative, compound5. Hemoglobin can be activated toward coupling reactions by reacting itwith γ-maleimidobutyric acid N-hydroxysuccinimide ester. Compound 5 canthen be covalently attached to the activated hemoglobin through itsmaleimide functionality. The adduct, 6, can generate NO steadily overseveral days in pH 7.4 phosphate buffer at 37° C. This property canalleviate side effects of underivatized blood substitutes, for example.

Nitric oxide synthase (NOS) working in conjunction with Hb can reload NOonto the hemes, thus a composition comprising NOS and Hb, or NOSconjugated to Hb can facilitate delivery of NO to the tissues. NOS ofneurons is preferable for this composition because the neuronal NOSresponds to oxygen tension. At low oxygen tension, the neuronal NOSproduces more NO; at high oxygen tension, NOS produces less NO. Thisform of NOS will efficiently reload NO onto the heme when Hb isdeoxygenated. NOS-Hb conjugates can be used when a blood substitute isindicated, and especially when an ischemic injury or condition ispresent.

Biologically compatible electron acceptors, are well known in the artand include, but are not limited to, superoxide dismutase and theoxidized forms of nicotinamide adenine dinucleotide (NAD⁺), nicotinamideadenine dinucleotide phosphate (NADP⁺), flavin adenine dinucleotide(FAD), flavin mononucleotide (FMN), ascorbate, dehydroascorbate andnitroxide spin traps. One or more electron acceptors can be conjugatedto Hb molecules, and can facilitate the conversion of thenitrosyl-Hb-electron acceptor form to the SNO-Hb-electron acceptor formby accepting the electron lost by NO in its transfer, in the form of NO⁺or as RSNO., to a β93Cys thiol group.

Nitroxides are one such class of electron acceptors which also act asfree radical scavengers. Nitroxides are stable free radicals that havebeen shown to have antioxidant catalytic activities which mimic those ofsuperoxide dismutase (SOD), and which when existing in vivo, caninteract with other substances to perform catalase-mimic activity.Nitroxides have been covalently attached to hemoglobin. See Hsia, J-C.,U.S. Pat. No. 5,591,710, the contents of which are incorporated byreference in their entirety. See also Liebmann, J. et al., Life Sci.54:503-509 (1994), describing nitroxide-conjugated bovine serum albuminand differential nitroxide concentrations among the different organs ofmice tested with the conjugate.

Methods for chemically attaching superoxide dismutase (SOD) to Hb areknown in the art. For example, see Quebec, E. A. and T. M. Chang, Artif.Cells Blood Substit. Immobil. Biotechnol. 23:693-705 (1995) andD'Agnillo, F. and Chang, T. M., Biomater. Artif. Cells ImmobilizationBiotechnol. 21:609-621 (1993). SOD attached to nitrosylhemoglobin candrive the reaction in which NO is transfered from the heme to thiol, byserving as an electron acceptor.

Like NO, CO is known to have vasodilator effects. (See Zakhary, R. etal., Proc. Natl. Acad. Sci USA 93:795-798 (1996).) A solution ofdeoxyhemoglobin can be derivatized with CO by exposing it to purified COgas in solution, until the desired extent of CO-bound Hb is reached.CO-derivatized Hb can be administered as a blood substitute orco-administered with other heme-based blood substitutes to alleviate theeffects (e.g., hypertension, intestinal pain and immobility) ofunderivatized hemoglobin. Hemoglobins can be derivatized to the extentnecessary to overcome constrictor effects, for example to a ratio ofCO/Hb in the range of approximately 0.1% to 10%.

Because the a subunits lack thiol groups to serve as NO⁺ acceptors fromthe heme, a blood substitute comprising α chains, for example in theform of dimers or tetramers, can be made which has different propertiesfrom a blood substitute comprising β chains alone, or comprising acombination of α and β chains. A blood substitute comprising a chains ofhemoglobin can be administered to an animal or to a human patient toalleviate a condition characterized by the effects caused by NO, forexample, in hypotensive shock.

β chains, unlike a chains, serve as active donors of NO to the tissues,rather than traps for NO. A blood substitute comprising β chains, forexample in the form of β diners or tetramers, can be made. Such a bloodsubstitute can be administered to a mammal to treat diseases or medicaldisorders wherein it is desired to deliver oxygen as well as NO or itsbiological equivalent to tissues affected by the disease, for example,in angina and other ischemic conditions.

Methods are known by which hemoglobin can be separated into its α and βsubunits and reconstituted. Separated, heme-free, alpha- andbeta-globins have been prepared from the heme-containing alpha and betasubunits of hemoglobin. (Yip, Y. K. et al., J. Biol. Chem. 247:7237-7244(1972)). Native human hemoglobin has been fully reconstituted fromseparated heme-free alpha and beta globin and from hemin. Preferably,heme is first added to the alpha-globin subunit. The heme-bound alphaglobin is them complexed to the heme-free beta subunit. Finally, heme isadded to the half-filled globin dimer, and tetrameric hemoglobin isobtained (Yip, Y. K. et al., Proc. Natl. Acad. Sci. USA 74;64-68(1997)).

The human alpha and beta globin genes reside on chromosomes 16 and 11,respectively. Both genes have been cloned and sequenced, (Liebhaber, etal., Proc. Natl. Acad. Sci. USA 77:7054-7058 (1980) (alpha-globingenomic DNA); Marotta, et al., J. Biol. Chem. 252:5040-5053 (1977) (betaglobin cDNA); Lawn, et al., Cell 21:647 (1980) (beta globin genomicDNA)).

Recombinant methods are available for the production of separate α and βsubunits of hemoglobin. For instance, Nagai and Thorgerson, (Nature309:810-812 (1984)) expressed in E. coli a hybrid protein consisting ofthe 31 amino terminal residues of the lambda cII protein, an,IIe-Glu-Gly-Arg linker, and the complete human beta globin chain. Theycleaved the hybrid immediately after the linker with blood coagulationfactor Xa, thus liberating the beta-globin chain. Later, (Nagai, K. etal., Proc. Natl. Acad. Sci. USA 82:7252-7255 (1985)) took therecombinant DNA-derived human beta globin, naturally derived human alphaglobin, and a source of heme and succeeded in producing active humanhemoglobin.

An efficient bacterial expression system for human alpha globin wasreported. (GB 8711614, filed May 16, 1987; see also WO 88/09179). Thisled to the production of wholly synthetic human hemoglobin by separateexpression of the insoluble globin subunits in separate bacterial celllines, and in situ refolding of the chains in the presence of oxidizedheme cofactor to obtain tetameric hemoglobin. A synthetic humanhemoglobin has been produced in yeast cells (EP 700997A1, filing dateOct. 5, 1990).

The properties of hemoglobin have been altered by specificallychemically crosslinking the alpha chains between the Lys99 of alpha 1and the Lys99 of alpha 2. (Walder, U.S. Pat. Nos. 4,600,531 and4,598,064; Snyder, et al., Proc. Natl. Acad. Sci USA 84:84 7280-7284(1987); Chaterjee, et al., J. Biol. Chem. 261:9927-9937 (1986)). Thischemical crosslinking was accomplished by reacting bis(3,5-dibromosalicyl) fumarate with deoxyhemoglobin A in the presence ofinositol hexaphosphate. The beta chains have also been chemicallycrosslinked. (Kavanaugh, M. P. et al., Biochemistry 27:1804-1808(1988)). Such linking methods or other suitable methods can be adaptedto methods of producing α or β dimers or other multimers, or for thecrosslinking of other polypeptides to the α and β chains. (For furthermethods to derivatize proteins and to conjugate proteins, see Hermansoh,G. T., Bioconjugate Techniques, Academic Press, 1996.)

The term hemoglobin or Hb as used herein includes variant forms such asnatural or artificial mutant forms differing by the addition, deletionand/or substitution of one or more contiguous or non-contiguous aminoacid residues, or modified polypeptides in which one or more residues ismodified, and mutants comprising one or more modified amino acidresidues. Hb also includes chemically modified forms as well asgenetically altered forms, such as fusion proteins, and truncated forms.It also includes Hbs of all animal species and variant forms thereof.The biological and/or chemical properties of these variant Hbs can bedifferent from those of hemoglobins which are found naturally occurringin animals.

It will be appreciated that NO exists in biological systems not only asnitric oxide gas, but also in various redox forms and as biologicallyactive adducts of nitric oxide such as S-nitrosothiols, which caninclude S-nitrosoproteins, S-nitroso-amino acids and otherS-nitrosothiols (Stamler, J. S. Cell 78:931-936 (1994)).

A blood substitute can be a biologically compatible liquid whichperforms one or more functions of naturally occurring blood found in amammal, such as oxygen carrying and/or delivery, NO carrying and/ordelivery, and the scavenging of free radicals. A blood substitute canalso comprise one or more components of such a liquid which, wheninfused into a mammal, perform one or more functions of naturallyoccurring blood. Examples of blood substitutes include preparations ofvarious forms of hemoglobin. Such preparations can also include otherbiologically active components, such as a low molecular weight thiol,nitrosothiol or NO donating agents, to allow transnitrosation. Lowmolecular weight thiols (i.e., relative to proteins and other biologicalmacromolecules) can include glutathione, cysteine, N-acetylcysteine,S-nitrosocysteinylglycine, S-nitrosocysteine, and S-nitrosohomocysteine.

The compounds and therapeutic preparations of this invention to be usedin medical treatment are intended to be used in therapeuticallyeffective amounts, in suitable compositions, which can be determined byone of skill in the art. Modes of administration are those known in theart which are most suitable to the affected site or system of themedical disorder. Intravenous infusion is a preferred mode ofadministration of various forms of hemoglobin to be used as a bloodsubstitute. Suitable compositions can include carriers, stabilizers orinert ingredients known to those of skill in the art, along withbiologically active component(s).

The term “therapeutically effective amount,” for the purposes of theinvention, refers to the amount of modified Hb and/or nitrosating agentwhich is effective to achieve its intended purpose. While individualneeds vary, determination of optimal ranges for effective amounts ofeach compound to be administered is within the skill of one in the art.Research animals such as dogs, baboons or rats can be used to determinedosages. Generally, dosages required to provide effective amounts of thecomposition or preparation, and which can be adjusted by one of ordinaryskill in the art, will vary, depending on the age, health, physicalcondition, sex, weight, extent of disease of the recipient, frequency oftreatment and the nature and scope of the desired effect. Dosages for aparticular patient can be determined by one of ordinary skill in the artusing conventional considerations, (e.g. by means of an appropriate,conventional pharmacological protocol). For example, dose responseexperiments for determining an appropriate dose of a heme-based bloodsubstitute can be performed to determine dosages necessary to produce aphysiological concentration of approximately 1 nM to 100 μM heme.Suitable pharmaceutical carriers or vehicles can be combined with activeingredients employed in a therapeutic composition, if necessary.

The present invention is further and more specifically illustrated inthe following examples, which are not intended to be limiting in anyway.

Exemplification

Materials and Methods for Assays

Determination of R—S—NO Concentration The concentration of R—S—NO groupsin a sample is based on the method reported in Saville, Analyst83:670-672 (1958). The quantification of the NO group, displaced fromthe thiol by mercuric ion, forms the basis of this highly sensitivemethod. The detection limit is in the range of 0.1-0.5 μM.2RSNO+Hg ²⁺→Hg(RS)₂+2NO⁻  (5)NO⁺+Ar—NH₂→Ar—N₂ ⁺+H₂O  (6)Ar′+Ar—N₂ ⁺→Ar—N═N—Ar′  (7)As shown (equations 5-7), the reaction proceeds in two steps. First, NO⁺is displaced from the RSNO by mercuric ion and reacts, under acidicconditions, with sulfanilamide (Ar—NH2). In a second step, the diazoniumsalt (which is formed in amounts equivalent to the thionitrite) is thencoupled with the aromatic amine, N-(1-naphthyl)-ethylenediamine (Ar′),to form an intensely colored azo dye which can be measured at 540 nm(ε˜50,000 M⁻¹ cm⁻¹). The same assay performed with the mercuric saltomitted allows for the simultaneous detection of nitrite. In principle,the second part of the Saville procedure is analogous to the classicalGriess reaction for the detection of nitrite.

The procedure is as follows:

-   Solution A: sulfanilamide 1% dissolved in 0.5 M HCl.-   Solution B: same solution as used in A to which 0.2% HgCl₂-   Solution C: 0.02% solution of N-(1-naphthyl)—ethylenediamine    dihydrochloride dissolved in 0.5 M HCl.

A given volume (50 μl-1 ml) of the sample to be assayed is added to anequivalent volume of solution A and solution B. The two samples are setaside for 5 minutes to allow formation of the diazonium salt, afterwhich an equivalent volume of solution C is added to each mixture. Colorformation, indicative of the azo dye product, is usually complete by 5minutes. The sample absorbance is then read spectrophotometrically at540 nm. The RSNO is quantified as the difference in absorbance betweensolution B and A. (i.e. B-A). In the event that the background nitriteconcentration is high (i.e. increased background in A), the accuracy ofthe measurement can be increased by the addition of an equivalent volumeof 0.5% ammonium sulfamate in acid (45 mM) 5 minutes prior to theaddition of sulfanilamide. The nitrous acid in solution reactsimmediately with excess ammonium sulfamate to form nitrogen gas andsulfate.

Concentrations of thiol greater than 500 μM in samples may interferewith the assay if nitrite is also present at micromolar concentration.Because nitrite will nitrosate indiscriminantly under the acidicconditions employed, thiols will effectively compete for reaction withsulfanilamide (present at 50 mM in this assay) as their concentrationapproaches the millimolar range. This will lead to artifactual detectionof RSNO. The problem can be avoided by (1) keeping the ratio of thiol tosulfanilamide <0.01, (2) first alkylating thiols in the solution, or (3)adding free thiols to standards to correct for the potential artifact.

Assay for S-nitrosohemoglobin and Nitrosyl(FeII)-Hemoglobin

A highly sensitive photolysis-chemiluminescence methodology wasemployed, which had been used for measuring RSNOs (S-nitrosothiols) inbiological systems (Gaston, B., et al., Proc. Natl. Acad. Sci. USA90:10957-10961 (1993); Stamler, J. S., et al., Proc. Natl. Acad. Sci USA89:7674-7677 (1992)). The method involves photolytic liberation of NOfrom the thiol, which is then detected in a chemiluminesencespectrometer by reaction with ozone. The same principle of operation canbe used to cleave (and measure) NO from nitrosyl-metal compounds(Antonini, E. and Brunori, M. In Hemoglobin and Myoglobin in TheirReactions with Ligands, American Elsevier Publishing Co., Inc., NewYork, pp. 29-31 (1971)). With adjustment of flow rates in the photolysiscell, complete photolysis of the NO ligand of Hb(FeII)NO is achieved.Standard curves derived from synthetic preparations of SNO-Hb,Hb(FeII)NO, and S-nitrosoglutathione were linear (R>0.99), virtuallysuperimposable, and revealing of sensitivity limits of approximately 1nM. Two analytical criteria were then found to reliably distinguishSNO-Hb from Hb(FeII)NO: 1) signals from SNO-Hb were eliminated bypretreatment of samples with 10-fold excess HgCl₂, while Hb(FeII)NO wasresistant to mercury challenge; and 2) treatment of SNO-Hb with HgCl₂produced nitrite (by standard Griess reactions) in quantitative yields,whereas similar treatment of Hb(FeII)NO did not. UV/VIS spectroscopyconfirmed that NO remained attached to heme in the presence of excessHgCl₂.

We linked a photolysis cell directly to the reaction chamber anddetector portion (bypassing the pyrolyzer) of a chemiluminescenceapparatus (model 543 thermal energy analyzer, Thermedix, Woburn Mass.).A sample (5 to 100 μl) is either introduced directly or introduced as achromatographic effluent from an attached high-performance liquid or gaschromatography system into the photolysis cell (Nitrolite, Thermedix,Woburn Mass.). This cell consists of a borosilicate glass coil (3 m×0.64cm o.d.×1 mm i.d., turned to a diameter of 6 cm and a width of 12 cm).The sample is introduced with a purge stream of helium (5 liters/min)and then irradiated with a 200-W mercury-vapor lamp (vertically mountedin the center of the photolysis coil on Teflon towers). The effluentfrom the photolysis coil is directed to a series of cold traps, whereliquid and gaseous fractions less volatile than nitric oxide (such asnitrite and nitrate) are removed. Nitric oxide is then carried by thehelium stream into the chemiluminescence spectrometer, in which freenitric oxide is detected by reaction with ozone. Signals are recorded ona digital integrator (model 3393A, Hewlett-Packard). Flow rates andillumination levels in the photolysis cell were designed to result incomplete photolysis of the S—N bond of S-nitrosothiols, as confirmed byanalysis of effluent from the cell according to the method of Saville(Saville, B., Analyst 83:670-672 (1958)).

To determine what fraction of the total nitric oxide detected in sampleswas derived from S-nitrosothiols, several control measurements wereperformed. Mercuric ion was used to displace nitric oxide selectivelyfrom the S-nitrosothiols (Saville, B., Analyst 83:670-672 (1958)).Comparison of measured nitric oxide concentrations from samplesalternatively pretreated or not pretreated with HgCl₂ ensured thatnitric oxide obtained by photolysis was derived specifically fromS-nitrosothiols. Similarly, as an added measure of confirmation, wedistinguished between S-nitrosothiols and free nitric oxide by comparingnitric oxide concentrations in samples alternatively exposed or notexposed to photolyzing illumination.

Methods for Spectrophotometric Experiments and NitrosylhemobloginFormation, Examples 14-20

Purified human HbA₀ was obtained from Apex Biosciences (Antonini, E. andBrunori, M. In Hemoglobin and Myoglobin in Their Reactions with Ligands,American Elsevier Publishing Co., Inc., New York (1971)). Thespectrophotometer used was a Perkin Elmer UV/vis Spectrometer Lambda 2S.All measurements were made at 23° C. in a sealed quartz cuvette to whichall additions were made. Deoxygenation was achieved by argon passagethrough a Hb solution within a sealed quartz cuvette. The degree ofdeoxygenation can be measured by UV/vis spectrum. Nitrosylation of hemesis achieved by addition of purified NO gas to deoxyHb and the productsquantitated by the extinction coefficient per Antonini and Brunori,supra.

EXAMPLE 1 Interactions of NO and RSNO with Hb

It was observed that naturally occurring N-oxides, such as NO and RSNOs(Gaston, B., et al., Proc. Natl. Acad. Sci. USA 90:10957-10961 (1993);Scharfstein, J. S., et al., J. Clin. Invest., 94:1432-1439 (1994);Clancy, R. M., et al., Proc. Natl. Acad. Sci. USA 91:3680-3684 (1994)),differed markedly in their reactions with Hb. NO bound very rapidly todeoxyHb (Hb[FeII]), forming relatively stable Hb[FeII]NO complexes (FIG.1A), and converted oxyHb (Hb[FeII]O₂) to methemoglobin (Hb[FeIII]) andnitrate (FIG. 1B), confirming previous reports (Olson, J. S., Methods inEnzymol. 76:631-651 (1981); Toothill, C., Brit. J. Anaesthy. 39:405-412(1967)). In contrast, RSNOs were found to participate intransnitrosation reactions with sulfhydryl groups of Hb, formingS-nitrosohemoglobin (SNO-Hb), and did not react with the heme centers ofeither deoxyHb or Hb(FeII) O₂ (FIGS. 1C and 1D).

A. Interaction of NO with deoxyHb

Conversion of deoxyhb (Hb[FeII]) to Hb(FeII)NO is observed uponincubation of Hb(FeII) with increasing concentrations of nitric oxide.See FIG. 1A. a. Deoxy Hb. b, c, d. Reaction mixtures of NO and Hb(FeII)in ratios of 1:1, 2:1 and 10:1, respectively. The reaction productHb(FeII)NO formed essentially instantaneously on addition of NO (i.e.within instrument dead time).

B. Interaction of NO with oxyHb

Conversion of oxyHb (Hb[Fe[II]O₂) to metHb (HbFe[III]) is observed uponincubation of oxyHb with increasing concentrations of NO. See FIG. 1B.a. oxy Hb. b, c, d. Reaction mixtures containing NO and oxyHb in ratiosof 1:1, 2:1 and 10:1, respectively. Methemoglobin formation occurredinstantaneously on addition of NO (i.e. within instrument dead time).

C. Interaction of S-nitrosothiols with deoxyhb

Conversion of Hb(FeII) to SNO-Hb(FeII) is observed upon incubation ofeither GSNO (shown) or S-nitrosocysteine (CYSNO) with deoxy Hb. There islittle (if any) interaction of RSNO with the heme functionalities of Hb.See FIG. 1C. a. deoxyHb. b, c, d. Reaction mixtures of GSNO and Hb(FeII)in ratios of 1:1, 2:1 and 10:1, respectively. Spectra were taken after60 min of incubation in b, c, and 15 min in d. Further analysis ofreaction products revealed the formation of moderate amounts of SNO-Hbin all cases. Yields of SNO-Hb (S—NO/Hb) in b, c, and d at 60 min were2.5%, 5% and 18.5%, respectively. (See FIG. 1D and FIG. 2A.)

D. Interaction of S-nitrosothiols with oxyHb

Conversion of Hb(FeII)O₂ to SNO-Hb(FeII)O₂ is observed upon incubationof either GSNO (shown) or CYSNO with oxyHb. There is little (if any)reaction of GSNO (or CYSNO) at the heme centers of Hb(FeII)O₂.Specifically, the capacity for O₂ binding to heme is unaffected byRSNOs. See FIG. 1D. a. oxyHb. b, c, d. Reaction mixtures of GSNO andoxyHb in ratios of 1:1, 2:1, and 10:1, respectively. Spectra were takenafter 60 min of incubation in the spectrophotometer. Further analysis ofreaction products revealed the formation of SNO-Hb in all cases. Yieldsof SNO-Hb in spectra b, c and d were 5%, 10% and 50% (S—NO/Hb),respectively. In 5 other determinations, the yield of S—NO/Hb was0.37±0.06 using GSNO (pH 7.4, 10-fold excess over Hb) and ˜2SNO/tetramer (1.97±0.06) using CYSNO (vida infra). These last data arein agreement with reports that human HbA contains 2 titratable SHgroups.

Methods

Human HbA₀ was purified from red cells as previously described(Kilbourn, R. G., et al., Biochem. Biophys. Res. Comm., 199:155-162(1994)). Nitric oxide solutions were rigorously degassed and purifiedaccording to standard procedure (Beckman, J. S., et al., Methods inNitric Oxide Research, Feelisch and Stamler, eds., Wiley Chichester,U.K. (1996)) and saturated solutions were transferred in air tightsyringes. Deoxygenation of Hb was achieved by addition of excessdithionite (No studies) or by reduction of Hb(FeII)O₂ through evacuationin Thunberg tubes (RSNO studies; as RSNOs react with dithionite). RSNOswere synthesized as previously described (Gaston, B., et al., (1993);Arnelle, D. R. and Stamler, J. S., Arch. Biochem. Biophys. 318:270-285(1995)) Incubations with HbA₀ were made in phosphate buffer, pH 7.4, 0.5mM EDTA. Quantifications of SNO-Hb were made according to the method ofSaville (Gaston, B., et al., (1993); Stamler, J. S., et al., Proc. Natl.Acad. Sci. USA, 90:444-448 (1992)) after purification of protein withSephadex G-25 columns. The Saville method, which assays free NO_(x) insolution, involves a diazotization reaction with sulfanilamide andsubsequent coupling with the chromophore N-(naphthyl)ethylenediamine. Nolow molecular weight S—NO complexes survived this purification and allactivity was protein precipitable. The reactions and spectra werecarried out using a Perkin Elmer UV/Vis Spectrometer, Lambda 2S.

EXAMPLE 2 Allosteric Function of O₂ in Regulation of Hb S-Nitrosylation

Oxygenation of Hb is associated with conformational changes thatincrease the reactivity of cysβ93 to alkylating reagents (Garel, C., etal., J. Biochem., 123:513-519 (1982); Jocelyn, P. C., Biochemistry ofthe SH Group, Academic Press, London, p.243 (1972); Craescu, C. T., etal., J. Biol. Chem., 261:14710-14716 (1986)). The physiologicalimportance of this effect has not been explained previously. It wasobserved here that rates of S-nitrosation of Hb were markedly dependenton conformational state. In the oxy conformation (R state),S-nitrosation was more rapid than in the deoxy conformation (T state)(FIG. 2A). The rate of S-nitrosation was accelerated in bothconformations by alkaline conditions (i.e., rate at pH 9.2>pH 7.4),which tends to expose the cysβ93 that is otherwise screened fromreaction by the C-terminal histidine 146β. The salt bridge (asp β94—hisβ146) tying down the histidine residue is loosened at high pH. Thesedata suggest that the increase in thiol reactivity associated with the Rstate derives, at least in part, from improved NO access rather than aconformation-induced change in pK.

A. Oxygenation Accelerates S-nitrosylation of Hb.

Rates of Hb S-nitrosation by S-nitrosocysteine (CYSNO) are faster in theoxy conformation (Hb[FeII]O₂) than in the deoxy state (Hb[FeII]).

Methods

Incubations were performed using 10-fold excess CYSNO over protein (50μM) in aerated 2% borate, 0.5 mM EDTA (oxyHb), or in a tonometer afterrapid O₂ evacuation (deoxyHb). At times shown in FIG. 2A, samples wererapidly desalted across G-25 columns (preequilibrated with phosphatebuffered saline, 0.5 mM EDTA, pH 7.4) to remove CYSNO, and analyzed forSNO-Hb by the method of Saville (Stamler, J. S., et al., Proc. Natl.Acad. Sci. USA, 89:444-448 (1992)).

B. Deoxygenation Accelerates Denitrosylation of Hb

Rates of RSNO decomposition (and transfer) are much faster in the deoxyconformation [SNO-Hb(FeII)] than in the oxy state [SNO-Hb(FeII)O₂]. Thedecomposition of SNO-Hb(FeII) is further accelerated by the presence ofexcess glutathione. Within the dead time of measurements according tothis method (˜15 seconds), a major fraction of SNO-Hb(FeII) wasconverted to GSNO.

Methods

Hbs in PBS (0.5 mM EDTA, pH 7.4) were incubated in air (oxy) or in atonometer previously evacuated of O₂ (deoxy). SNO-Hb(FeII)O₂decomposition was determined by the method of Saville (Saville, B.,Analyst 83:670-672 (1958)). Spontaneous decomposition of SNO-Hb(FeII)was followed spectrophotometrically by formation of Hb(FeII)NO.Transnitrosation reactions with glutathione were performed by additionof 100-fold excess glutathione over protein (50 μM), immediateprocessing of the reaction mixture under anaerobic conditions followedby rapid TCA precipitation, and analysis of RSNO in the supernatant.Rates of NO group transfer were too rapid to measure accurately by thestandard methods used in this study.

EXAMPLE 3 NO-Related Interactions with Cysteine Residues of Hb inPhysiological Systems

Given that Hb is largely contained within red blood cells, potentialmechanisms by which S-nitrosation of intracellular protein might occurwere explored. Incubation of oxygenated rat red blood cells withS-nitrosocysteine resulted in very rapid formation of intracellularSNO-Hb(FeII)O₂ (FIG. 3A). Rapid oxidation of Hb was not observed underthese conditions. Intraerythrocytic SNO-Hb also formed when red bloodcells were treated with S-nitrosohomocysteine orS-nitrosocysteinylglycine, but not with S-nitrosoglutathione (GSNO).Thus, erythrocyte access of RSNOs is thiol group specific. Exposure ofoxygenated red blood cells to NO resulted primarily in metHb formation.

Endothelium-Derived Relaxing Factor (EDRF) and Hemoglobin

Hb-mediated inhibition of endothelium-dependent relaxations is commonlyused as a marker of NO responses. Inasmuch as reactions with eithermetal or thiol centers of Hb should lead to attenuated NO/EDRF(endothelium-derived relaxing factor) responses, experiments wereperformed to elucidate the molecular basis of inhibition. Hbpreparations in which β93 thiol groups had been blocked withN-ethylmaleimide (NEM) or the hemes blocked by cyanmet(FeIIICN)-derivitization were studied in an aortic ring bioassay, andtheir activities were compared with that of native Hb. Both cyanmet-Hband NEM-Hb caused increases in vessel tone and attenuated acetylcholine(EDRF)-mediated relaxations (FIG. 3B). However, native Hb wassignificantly more effective than either of the modified Hb preparations(FIG. 3B). Taken in aggregate, these studies suggest that both the thioland metal groups of Hb contribute to its NO-related activity. To verifyformation of an S-nitrosothiol in Hb, a bioassay was used in which 2 cmsegments of thoracic aorta were interposed in Tygon tubing, throughwhich 3 cc of Krebs solution containing Hb (4 μM) and ACh (2 μM) werecirculated by roller pump (1.5 cc/min×5 min). Analysis of the effluent(Gaston, B., et al., (1993)) revealed the formation of SNO-Hb (20±4 nM)in 5 of 5 experiments.

A. S-nitrosation of Intraervthrocytic Hb

Incubation of rat erythrocytes with S-nitrosocysteine (equimolar to heme(5 mM); phosphate buffer pH 7.4, 25° C.) leads to rapid formation ofintracellular SNO-Hb(FeII)O₂. MetHb does not form rapidly. Separation ofintracellular RSNOs across G-25 columns reveals that only a smallpercentage exists as low molecular weight S-nitrosothiol (e.g. GSNO) atmost time points. By 60 min, 3 of the 4 available SH groups of Hb wereS-nitrosated (note that rat Hb contains 4 reactive SH groups). See FIG.3A. Inset shows spectra of SNO-Hb isolated from rat erythrocytes andrelated analyses. Spectrum A is that of SNO-Hb isolated fromerythrocytes following G-25 chromatography. Treatment of A withdithionite results in reduction of the S—NO moiety, liberating free NOwhich is autocaptured by deoxy Hb, forming Hb(FeII)NO (note thatdithionite simultaneously deoxygenates Hb) (spectrum C). This spectrum(C) reveals a stoichiometry of ˜3 S—NOs per tetramer. The spectrum ofHb(FeII)NO containing 4 NO's per tetramer is shown for comparison(inset, spectrum B).

Methods

At shown intervals, red blood cells were pelleted rapidly bycentrifugation, washed three times, lysed in deionized water at 4° C.,and the cytosolic fraction subjected to rapid desalting across G-25columns. Intracellular SNO-Hb was measured by the method of Saville(Gaston, B., et al., (1992); Stamler, J. S., et al., Proc. Natl. Acad.Sci. USA, 89:444-448 (1992)), and confirmed spectroscopically (inset ofFIG. 3A) as described above.

B. Molecular Basis of EDRF/Hb Interaction.

The effects of native Hb on EDRF responses were compared with Hbpreparations in which the thiol or heme centers had been blocked byalkylation or cyanmet derivitization, respectively. All preparations ofHb elicited contractions; however, those of native Hb (in which both SHand metal centers are free for interaction) were most pronounced. SeeFIG. 3B. Likewise, acetylcholine (ACh) mediated relaxations were mosteffectively inhibited by native Hb. Relaxations were inhibited to lesserdegrees by cyanmet Hb (CN-Hb)(in which hemes were blocked from reaction)and NEM-Hb (in which thiol groups were alkylated by N-ethylmaleimide).See Table 1. These data illustrate that both heme and β93SH groups of Hbcontribute to reversal of EDRF responses. Direct measurement of SNO-Hb,formed from EDRF under similar conditions, is described in Example 8.

Methods

Descending rabbit thoracic aorta were cut into 3 mm rings and mounted onstirrups attached to force transducers (model FT03, Grass Instruments,Quincy, Mass.) for measurement of isometric tone. The details of thisbioassay system have been previously described (Stamler, J. S., et al.,Proc. Natl. Acad. Sci. USA, 89:444-448 (1992)). Cyanmet Hb was preparedfrom human HbA according to published protocols (Kilbourn, R. G. et al.Biochem. Biophys. Res. Comm., 199:155-162, (1994)). Alkylation of HbAwith N-ethylmaleimide was followed by desalting across G-25 Sephadex toremove excess NEM. Removal of unmodified Hbcysβ93 was achieved bypassage through Hg-containing affinity columns. The alkylation of freeSH groups was verified using 5,5′-dithio-bis[2-nitrobenzoic acid].

TABLE 1 % INCREASE IN ADDITIONS TENSION % ACh RELAXATION Hb (1 μM) 40.8± 2.3 (n = 7) 31.9 ± 6.9 (n = 7) NEM-Hb (1 μM) 29.4 ± 1.3** (n = 7) 60.5± 3.9* (n = 7) CN-Hb (1 μM) 12.9 ± 3.0** (n = 6) 80.7 ± 1.0**† (n = 4)ACh (1 μM) 98.3 ± 0.6 (n = 10) *P < 0.01; **P < 0.001, Compared to Hb;†P < 0.001, Compared to ACh

EXAMPLE 4 Transduction of SNO-Hb Vasoactivity

Arterial red blood cells contain two physiologically important forms ofhemoglobin: Hb(FeII)O₂ and Hb(FeIII) (Antonini, E. and Brunori, M. InHemoglobin and Myoglobin in Their Reactions with Ligands, AmericanElsevier Publishing Co., Inc., New York, pp. 29-31 (1971)).Arterial-venous differences in the S-nitrosothiol content ofintraerythrocytic Hb suggest that the NO group is released during redcell transit. Such findings raise the possibility of functionalconsequences, perhaps influenced by the redox state of heme and itsoccupation by ligand. SNO-Hb(FeII)O₂ was found to possess modest NO-likeactivity when tested in a vascular ring bioassay. Specifically, thecontraction elicited by SNO-Hb(FeII)O₂ was less than that of nativeHb(FeII)O₂, indicating that S-nitrosation partially reverses thecontractile effects of Hb (FIG. 4A). By comparison, SNO-Hb(FeIII) wasfound to be a vasodilator (FIG. 4A). Notably, free NO was devoid ofrelaxant activity in the presence of Hb(FeII)O₂ or Hb(FeIII).

Red blood cells contain millimolar concentrations of glutathione. Asequilibria among RSNOs are rapidly established through RSNO/thiolexchange (Arnelle, D. R. and Stamler, J. S., Arch. Biochem. Biophy.,318:279-285 (1995)), the vasoactivity of SNO-Hb was reassessed in thepresence of glutathione. FIG. 4B illustrates that glutathionepotentiated the vasodilator activity of both SNO-Hb(FeII)O₂ andSNO-Hb(FeIII). GSNO formation under these conditions (confirmedchemically and in bioassay experiments) appeared to fully account forthis effect. Further kinetic analyses revealed that transnitrosationinvolving glutathione was more strongly favored in the equilibrium withSNO-Hb(FeIII) than SNO-Hb(FeII)O₂ (FIG. 4C). Given the findings ofsteady-state levels of SNO-Hb in red blood cells (Table 2 and FIG. 3A),these results suggest that 1) the equilibrium between naturallyoccurring RSNOs and Hb(cysβ93) lies toward SNO-Hb under physiologicalconditions; 2) that transnitrosation reactions involving SNO-Hb and GSHare likely to occur within red blood cells (in these studies, lowmolecular weight RSNOs have been found in erythrocytes loaded withSNO-Hb); and 3) that oxidation of the metal center of Hb shift theequilibrium toward GSNO, thereby potentially influencing bioactivity.

Additional mechanisms of NO group release from SNO-Hb were sought.Arterial-venous differences in levels of SNO-Hb raised the possibilitythat S—NO bond stability may be regulated by the changes in Hbconformation accompanying deoxygenation. To test this possibility, therates of NO group release from SNO-Hb(FeII)O₂ and SNO-Hb(FeIII) werecompared. Deoxygenation was found to enhance the rate of SNO-Hbdecomposition (FIG. 2B). These rates were accelerated greatly byglutathione in a reaction yielding GSNO (FIG. 2B). The resultsillustrate that O₂-metal interactions influence S—NO affinity, andsuggest a new allosteric function for Hb.

For SNO-Hb to be of physiological importance it must transduce itsNO-related activity across the erythrocyte membrane. This possibilitywas explored by incubating erythrocytes containing SNO-Hb in physiologicbuffer, and measuring the accumulation of extracellular RSNOs over time.FIG. 4D illustrates that red blood cells export low molecular weight(trichloroacetic acid soluble) S-nitrosothiols under these conditions.Importantly, the degree of hemolysis in these experiments was trivial(<0.5%), and correction for lysis did not significantly impact on ratesof RSNO release. These results establish that an equilibrium existsbetween low molecular weight and protein RSNOs within the red cell, andthat intracellular location is unlikely to be a limiting factor in thetransduction of such NO-related activity to the vessel wall.

A. Concentration-Effect Responses of Different SNO-Hb Preparations

Contractile effects of Hb(FeII)O₂(▴) are shown to be partially reversedby S-nitrosation (SNO-Hb[FeII]O₂(▪); P=0.02 by ANOVA vs Hb(FeII)O₂) (SeeFIG. 4A.). Oxidation of the metal center of SNO-Hb (SNO-Hb[FeIII](●))converts the protein into a vasodilator (P<0.0001 by ANOVA vs.SNO-Hb[FeII]O₂), with potency comparable to that of otherS-nitrosoproteins (Stamler, J. S., et al., Proc. Natl. Acad. Sci. USA,89:444-448 (1992)). The contractile properties of Hb(FeIII) are shownfor comparison (□); n=6-17 for each data point.

Methods

Details of the vessel ring bioassay have been published (Stamler, J. S.,et al., Proc. Natl. Acad. Sci. USA 89:444-448 (1992)). SNO-Hb(FeII)O₂preparations were synthesized with 10-fold excess S-nitrosocysteine(CYSNO) over Hb(FeII)O₂ protein (2% borate, 0.5 mM EDTA, ˜15 minincubation), after which desalting was performed across Sephadex G-25columns. CYSNO was synthesized in 0.5 N HCl, 0.5 mM EDTA and thenneutralized (1:1) in 1 M phosphate buffer containing 0.5 mM EDTA.SNO-Hb(FeIII) preparations followed a similar protocol, but usedHb(FeIII) as starting material. The latter was synthesized by treatmentof Hb(FeII)O₂ with excess ferricyanide, followed by desalting acrossG-25 columns. SNO-Hb concentrations were verified spectroscopically andthe S-nitrosothiol content was determined by the method of Saville(Stamler, J. S., et al., Proc. Nat. Acad. Sci USA 89:444-448 (1992)).The S—NO/tetramer stoichiometry for both SNO-Hb preparations was ˜2.Oxidation of the heme was undetectable by uv-spectophotometric methods.

B. Potentiation of SNO-Hb Effects by Glutathione

Addition of glutathione (100 μM) to bioassay chambers potentiates thedose-response to both SNO-Hb(FeII)O₂(▪) and SNO-Hb(FeIII) (●) (See FIG.4B. n=6-12; p<0.0001 for both by ANOVA, compared with the respectivetracings in FIG. 4A. Glutathione had a transient affect on baseline tonein some experiments, and did not significantly influence the response toHb(FeII)O₂ (▴)

C. Transnitrosation Between SNO-Hb and Glutathione

Rates of NO group transfer from SNO-Hb (100 μM) to glutathione (10 mM)are displayed for SNO-Hb(FeII)O₂ (oxy) and SNO-Hb(FeIII) (met) (n=5).Data are presented as the amount of GSNO formed relative to the startingSNO-Hb concentration. The transfer is more rapid for SNO-Hb(FeIII) thanSNO-Hb(FeII)O₂ (p<0.002 by ANOVA), suggesting that the GSNO/SNO-Hbequilibrium is shifted toward GSNO by formation of metHb.

Methods

Thiol/SNO-Hb exchange, forming GSNO, was verified chemically (Stamler,J. S., et al., Proc. Natl. Acad. Sci. USA, 89:444-448 (1992)) followingtrichloroacetic acid precipitation (n=5). These results were verified inseparate experiments by measuring the residual SNO-Hb concentration,following separation of reaction mixtures across G-25 columns.

D. Export of S-nitrosothiols by Red Blood Cells

Human red blood cells containing SNO-Hb are shown to export lowmolecular weight RSNOs over time. Hemolysis, which ranged from 0−<0.5%over one hour and did not correlate with rates of RSNO release, couldaccount for only a trivial fraction of the measured extracellular RSNO.

Methods

Packed human red blood cells were obtained by centrifugation, washed,and resuspended in phosphate buffered saline containing 5 mM SNOCYS (0.5mM EDTA, pH 7.4) for one hour. This results in a red cell preparationcontaining SNO-Hb (FeIIO₂/FeIII mixture) with a stoichiometry of 0.5S—NO/tetramer. The red blood cells were then washed repeatedly to removeresidual CYSNO (verified), and incubated in Krebs' solution (1:4). Theaccumulation of extracellular RSNO was measured over time by the methodof Saville (Saville, B., Analyst, 83:670-672 (1958)). Hemolysis wasdetermined by spectral analysis of red blood cell supernatants followingcentrifugation.

EXAMPLE 5 SNO-Hb Bioactivity In Vivo

Systemic administration of cell-free Hb results in hypertensiveresponses which have been attributed to NO scavenging by the heme(Vogel, W. M., et al., Am. J. Physiol. 251:H413-H420 (1986); Olsen, S.B., et al., Circulation 93:329-332 (1996)). To determine if SNO-Hb isfree of this adverse affect, and to explore if in vitro mechanisms of NOrelease extend to the in vivo circumstance, we compared responses to Hband SNO-Hb infused as a bolus into the femoral vein of anesthetizedrats. As illustrated in FIG. 5, Hb(FeII)O₂ (200 nmol/kg) caused anincrease in mean arterial pressure of 20±3 mm Hg (n=4; P<0.05). Incontrast, SNO-Hb(FeII)O₂ did not exhibit hypertensive effects andSNO-Hb(FeIII) elicited hypotensive responses (FIG. 5). Thus, theprofiles of these compounds in vivo closely resemble those seen in vitro(FIG. 4A). Moreover, to demonstrate that the physiological responses ofred cells are comparable to those of cell-free Hb preparations,erythrocytes containing SNO-Hb were injected into the femoral vein ofrats pretreated with L-NMMA (50 mg/kg) to deplete endogenous RSNOs. Atlevels of SNO-Hb comparable to those found in the normal rat (0.1-0.5μM), SNO-Hb containing red blood cells elicited hypotensive responses(8+1 mm Hg; mean±SEM; n=9), whereas native (SNO-Hb depleted) red bloodcells did not (P=0.001). These changes in mean blood pressure of ˜10%are on the order of those that differentiate normotension fromhypertension in man, and in the therapeutic range of someantihypertensive regimens. The effects of both Hb and SNO-Hb—whethercell-free or contained within red cells—were transient, suggesting thatS-nitrosylation of Hb and metabolism of SNO-Hb is occurring in vivo,with consequent restoration of blood pressure. The bioactivity of SNO-Hbin blood, where S—NO/heme stoichiometries approach 1:50,000, is adramatic illustration of the resistance of this NO-related activity toHb(Fe) inactivation.

In vivo Effects of Cell-Free Hb and SNO-Hbs

Administration of 2-200 nmol/kg Hb(FeII)O₂ (as a bolus) into the femoralvein of a Sprague-Dawley rat is shown to increase mean arterial pressurein a dose-dependent manner. At 200 nmol/kg, mean arterial pressureincreased by 25 mm Hg (20±3 mm Hg; n=4; P<0.05). Elevations in bloodpressure reversed within 10-15 minutes. SNO-Hb(FeII)O₂ infusions (overthe same dose range) are shown to ameliorate Hb(FeII)O₂-inducedhypertension without causing overt changes in blood pressure. A similarresponse was seen at higher doses. By comparison, SNO-Hb(FeIII)infusions caused a significant fall in mean arterial pressure (pre 108±4mm Hg; post 74±6 mm Hg, n=5; P<0.05) at the highest dose (200 nmol/kg).Hypotensive responses tended to be transient with blood pressurenormalizing over 10 minutes. A fall in blood pressure was also seen withinjection of erythrocytes containing SNO-Hb.

Methods

Rats were anesthetized by intraperitoneal injection of pentobarbital andthe femoral arteries and veins accessed by local cut down. The arterywas then cannulated and the blood pressure monitored continuously usinga Viggo Spectramed pressure transducer attached to a Gould recorder. AnIBM PC (DATA Q Codas) was used for data acquisition.

EXAMPLE 6 Loading of Red Blood Cells with S-Nitrosothiols

Incubation of rat erythrocytes with S-nitrosocysteine (equimolar to heme(5 mM); phosphate buffer pH 7.4, 25° C.) leads to rapid formation ofintracellular S-nitrosothiols. MetHb does not form rapidly. Separationof cell content across G-25 columns establishes the formation ofintraerythrocytic low molecular weight S-nitrosothiol, e.g.S-nitrosoglutathione, (GSNO). By 2 minutes, one can achieve as much asmillimolar GSNO.

Method for Assay of RSNO

S-nitrosocysteine (5 mM) treated red blood cells are pelleted rapidly bycentrifugation, washed three times, lysed in deionized water at 4° C.,and the cytosolic fraction subjected to rapid desalting across G-25columns. Intracellular RSNO is measured by the method of Saville and canbe confirmed spectroscopically.

Effects on Blood Pressure from Loaded Red Blood Cells

Red blood cells treated with S-nitroscysteine (to produce SNO-RBCs) andintroduced into the femoral vein of a Sprague-Dawley rat decreased meanarterial pressure in a dose-dependent manner. For red blood cells inwhich SNO-Hb was assayed at 0.3 μM (the endogenous in vivo SNO-Hbconcentration), arterial pressure decreased by 8±1 mm Hg (mean±SEM for 9experiments; p<0.001 compared to untreated red blood cell controls). Forred blood cells in which SNO-Hb was assayed at 0.5 μM, arterial pressuredecreased by 10 mm Hg. For red blood cells in which SNO-Hb was assayedat 0.1 μM (a sub-endogenous SNO-Hb concentration), arterial pressuredecreased by 6 mm Hg. The administration of untreated red blood cellscaused no effect or a slight increase in arterial blood pressure.Administration of L-monomethyl-L-arginine (L-NMMA; 50 mg/kg) caused anincrease in blood pressure of about 20 mm Hg. Changes in blood pressurefrom a bolus administration of loaded red blood cells lasted 15-20minutes.

Further Methods

Rats were anesthetized by intraperitoneal injection of pentobarbital andthe femoral arteries and veins accessed by local cut down. The arterywas then cannulated and the blood pressure monitored continuously usinga Viggo Spectramed pressure transducer attached to a Gould recorder. AnIBM PC (DATA Q Codas) was used for data acquisition.

EXAMPLE 7 Effects of SNO-Hb on Coronary Vasodilation Coronary Flow andBlood Pressure

SNO-Hb was synthesized as described in Example 4A. Completion of thereaction was determined as described in Example 4A. Twenty-four healthymongrel dogs (25-30 kg) were anesthetized with intravenous thiamylalsodium (60-80 mg/kg) and subjected to left thoracotomy in the fourthintercostal space. The left circumflex coronary artery distal to theleft atrial appendage was minimally dissected. A pair of 7-MHzpiezoelectric crystals (1.5×2.5 mm, 15-20 mg) was attached to a Dacronbacking and sutured to the adventitia on opposite surfaces of thedissected vessel segment with 6-0 prolene. Oscilloscope monitoring andon-line sonomicrometry (sonomicrometer 120-2, Triton Technology, SanDiego, Calif.) were used to ensure proper crystal position. A pulseDoppler flow probe (10 MHz, cuff type) was implanted distal to thecrystals. An inflatable balloon occluder was also placed distal to theflow probe. All branches of the circumflex artery between the crystalsand the occluder were ligated. Heparin sodium-filled polyvinyl catheterswere inserted into the left ventricular cavity via the apex, into theleft atrium via the atrial appendage, and into the ascending aorta viathe left internal thoracic artery. The catheters, tubing, and wires weretunnelled to a subcutaneous pouch at the base of the neck.

After a 10 to 15 day recovery period, the catheters and wires wereexteriorized under general anesthesia, and 2-3 days later, each dog wasgiven a bolus injection of SNO-Hb (0.4 mg) to evaluate vascularresponse. Two dogs that demonstrated <5% dilation of epicardial coronaryvessels were excluded from subsequent studies, and two were excludedbecause of other technical reasons.

Dogs were trained and studied while loosely restrained and lying awakein the lateral recumbent position. The laboratory was kept dimlyilluminated and quiet. Aortic pressure, left ventricular end-diastolicpressure dP/dt external coronary diameter and coronary flow weremonitored continuously. In 10 dogs, 0.1 ml of SNO-Hb solution, 50 nM/kg,was injected via the left atrial catheter. To verify potential effectsof solvent on vasculature, 0.1 ml injections of 30% ethanol in distilledwater were given as vehicle control. Between injections, phasic coronaryblood flow and coronary artery diameter were allowed to return topreinjection levels (minimum 15 minutes). Allowing a 15 minute periodbetween injections resulted in no modification of repeated doesinjections. To assess the direct and potential flow mediated indirectvasodilation effects of SNO-Hb on the conductance vessels, the dose wasrepeated in 6 of 10 dogs with partial inflation of the adjustableoccluder to maintain coronary blood flow at or slightly belowpreinjection levels. The response to acetylcholine chloride (SigmaChemical) was assessed in another group of 10 dogs following a similarprotocol to that used for SNO-Hb.

Epicardial coronary diameter, coronary blood flow, heart rate, andaortic and left ventricular end-diagnostic pressures were comparedbefore and after each SNO-Hb injection. The maximum changes in coronarydimension and blood flow were expressed as a function of increasingdoses of SNO-Hb. The response of coronary dimension to increasing dosesfollowed a characteristic sigmoid dose-response curve that could bedescribed by the following equation${Effect} = \frac{{maximal}\quad{effect} \times {dose}}{K_{D} + {dose}}$where K_(D) is the drug-receptor complex dissociation constant and isthe dose at which 50% of the maximum response (EC₅₀) is achieved. Ineach animal, a nonlinear least-squares regression (r²>0.90) wasperformed on the dose-response data. The regression was constrained tothe above equation. From the regression, values for maximum response andK_(D) were obtained for each individual animal. The mean of these valueswas then calculated to obtain an average K_(D) and maximum response forthe study group. These values were used to generate a mean curve, whichwas plotted with the mean dose-response values. (See FIGS. 6A-6F.)

EXAMPLE 8 Endogenous Levels of S-Nitrosohemoglobin andNitrosyl(FeII)-Hemoglobin in Blood

To determine if SNO-Hb is naturally occurring in the blood, and if so,its relationship to the O₂ transport capacity and nitrosylated-hemecontent of red cells, an analytical approach was developed to assay theS-nitrosothiol and nitrosyl-heme content of erythrocytes (Table 2).Arterial blood was obtained from the left ventricle of anesthetized ratsby direct puncture and venous blood was obtained from the jugular veinand inferior vena cava. Hb was then purified from red cells and assayedfor RSNO and (FeII)NO content. Arterial blood contained significantlevels of SNO-Hb, whereas levels were virtually undetectable in venousblood (Table 2). Measurements made 45 minutes after infusion of the NOsynthase inhibitor N^(ω)-monomethyl-L-arginine (L-NMMA) (So mg/kg),showed a depletion of SNO-Hb as well as total Hb-NO (82 and 50±18%,respectively; n=3-5; p<0.05). These data establish the endogenous originof SNO-Hb, although some environmental contribution is not excluded. Thearterial-venous distribution seen for SNO-Hb was reversed in the case ofHb(FeII)NO, which was detected in higher concentrations in partiallydeoxygenated (venous) erythrocytes (Table 2). Accordingly, theproportion of nitrosylated protein thiol and heme appears to depend onthe oxygenation state of the blood. Consistent with these findings,Wennmalm and coworkers have shown that Hb(FeII)NO forms mainly in venous(partially deoxygenated) blood (Wennmalm, A., et al., Br. J. Pharmacol.106(3):507-508 (1992)). However, levels of Hb(FeII)NO in vivo aretypically too low to be detected (by EPR) and SNO-Hb is EPR-silent(i.e., it is not paramagnetic). Thus, photolysis-chemiluminesencerepresents an important technological advance, as it is the firstmethodology capable of making quantitative and functional assessments ofNO binding to Hb under normal physiological conditions.

TABLE 2 Endogenous Levels of S-Nitrosohemoglobin andNitrosyl(FeII)-Hemoglobin in Blood Site SNO-Hb (nM) Hb(FeI)NO (nm)Arterial 311 ± 55* 536 ± 99† Venous 32 ± 14 894 ± 126 *P < 0.05 vsvenous; †P < 0.05 for paired samples vs venousMethods

Blood was obtained from the left ventricle (arterial) and jugular vein(venous) of anesthetized Sprague-Dawley rats. Comparable venous valueswere obtained in blood from the inferior vena cava. Red blood cells wereisolated by centrifugation at 800 g, washed three times in phosphatebuffered saline at 4° C., lysed by the addition of 4-fold excess volumeof deionized water containing 0.5 mM EDTA, and desalted rapidly acrossG-25 columns according to the method of Penefsky at 4° C. In 24 rats, Hbsamples were divided in two aliquots which were then treated or nottreated with 10-fold excess HgCl₂ over protein concentration as measuredby the method of Bradford. Determinations of SNO-Hb and Hb(FeII)NO weremade by photolysis-chemiluminescence. In 12 additional rats, furtherverification of the presence of SNO-Hb was made by assaying for nitriteafter HgCl₂ treatment. Specifically, samples (with and without HgCl₂)were separated across Amicon-3 (Centricon filters, m.w. cut off 3,000)at 4° C. for 1 h, and the low molecular weight fractions collected inairtight syringes containing 1 μM glutathione in 0.5 N HCl. Under theseconditions, any nitrite present was converted to S-nitrosoglutathione,which was then measured by photolysis-chemiluminescence (detection limit˜1 nM). SNO-Hb was present in all arterial samples, and levelsdetermined by this method (286±33 nM) were virtually identical to andnot statistically different from those shown in Table 2. In venousblood, SNO-Hb was undetectable (0.00±25 nM); levels were notstatistically different from those given above.

Method for Assay of S-nitrosohemoglobin

A highly sensitive photolysis-chemiluminescence methodology wasemployed. A somewhat similar assay has been used for measuring RSNOs(S-nitrosothiols) in biological systems (Gaston, B., et al., Proc. Natl.Acad. Sci. USA 90:10957-10961 (1993); Stamler, J. S., et al., Proc.Natl. Acad. Sci. USA 89:7675-7677 (1992)). The method involvesphotolytic liberation of NO from the thiol, which is then detected in achemiluminesence spectrometer by reaction with ozone. The same principleof operation can be used to cleave (and measure) NO from nitrosyl-metalcompounds (Antonini, E. and Brunori, M. In Hemoglobin and Myoglobin inTheir Reactions with Ligands, American Elsevier Publishing Co., Inc.,New York, pp. 29-31 (1971)). With adjustment of flow rates in thephotolysis cell, complete photolysis of the NO ligand of Hb(FeII)NOcould be achieved. Standard curves derived from synthetic preparationsof SNO-Hb, Hb(FeII)NO, and S-nitrosoglutathione were linear (R>0.99),virtually superimposable, and revealing of sensitivity limits ofapproximately 1 nM. Two analytical criteria were then found to reliablydistinguish SNO-Hb from Hb(FeII)NO: 1) signals from SNO-Hb wereeliminated by pretreatment of samples with 10-fold excess HgCl₂, whileHb(FeII)NO was resistant to mercury challenge; and 2) treatment ofSNO-Hb with HgCl₂ produced nitrite (by standard Griess reactions) inquantitative yields, whereas similar treatment of Hb(FeII)NO did not.UV/VIS spectroscopy confirmed that NO remained attached to heme in thepresence of excess HgCl₂.

EXAMPLE 9 Inhibition of Platelet Aggregation by S-Nitrosohemoglobins

Methods to prepare human HbA₀ were as described in Example 1 “Methods”section. Methods to make SNO-Hb(FeII)O₂ were as described for Example2A. Methods to make SNO-Hb(FeIII) were as in Example 1 (see parts B, C,and “Methods” in Example 1). Quantifications of SNO-hemoglobins weremade as in Example 1 according to the method of Saville (Saville, B.,Analyst 83:670-672 (1958)) and by the assay as described in Example 8,“Method for assay of S-nitrosohemoglobin.”

Venous blood, anticoagulated with 3.4 nM sodium citrate, was obtainedfrom volunteers who had not consumed acetylsalicylic acid or any otherplatelet-active agent for at least 10 days. Platelet-rich plasma wasprepared by centrifugation at 150×g for 10 minutes at 25° C. and wasused within 2 hours of collection. Platelet counts were determined witha Coulter counter (model ZM) to be 1.5 to 3×10⁸/ml.

Aggregation of platelet-rich plasma was monitored by a standardnephelometric technique in which results have been shown to correlatewith bleeding times. Aliquots (0.3 ml) of platelets were incubated at37° C. and stirred at 1000 rpm in a PAP-4 aggregometer (Biodata,Hatsboro, PA). Hemoglobins were preincubated with platelets for 10 minand aggregations were induced with 5 μM ADP. Aggregations werequantified by measuring the maximal rate and extent of change of lighttransmittance and are expressed as a normalized value relative tocontrol aggregations performed in the absence of hemoglobin.

The results of the aggregation assays are shown in FIGS. 7A, 7B and 7C.Standard deviations are shown as vertical bars. SNO-Hb[Fe(II)]O₂ causessome inhibition of platelet aggregation at the higher concentrationstested. SNO-Hb[Fe(III)] also inhibits platelet aggregation when presentat concentrations of 1 μM and above, but to a much greater extent thanSNO-Hb[Fe(II)]O₂.

EXAMPLE 10 Effect of SNO-Hbs on cGMP

Platelet rich plasma (PRP) was incubated with either hemoglobin, SNO-oxyHb, or SNO-metHb for 5 min, after which the assay was terminated by theaddition of 0.5 ml of ice cold trichloroacetic acid to 10%. Etherextractions of the supernatant were performed to remove trichloroaceticacid, and acetylation of samples with acetic anhydride was used toincrease the sensitivity of the assay. Measurements of cyclic GMP wereperformed by radioimmunoassay (Stamler, J. et al., Circ. Res. 65:789-795(1989)).

Results are shown in FIG. 8. For all concentrations of Hb tested (1, 10and 100 μM), the concentration of cGMP measured for SNO-Hb(FeIII) wasless than that of native Hb.

EXAMPLE 11 Polynitrosation of Hb

-   A. HbA₀ (oxy) was incubated with S-nitrosoglutathione at a ratio of    6.25 S-nitrosoglutathione/HbA₀ for 240 minutes at pH 7.4 at 25° C.    and desalted over Sephadex G-25 columns. Spectra were run in the    presence (spectrum B, FIG. 9A) and absence (spectrum A, FIG. 9A) of    dithionite. The shift in the spectrum is indicative of 2 SNO    groups/tetramer.-   B. HbA₀ was incubated with 100-fold excess S-nitrosoglutathione over    protein for 240 minutes at pH 9.2, followed by desalting over a G-25    column. A portion was then treated with dithionite. The spectra in    FIG. 9B indicate that Hb has been nitrosated at multiple sites.-   C. HbA₀ was treated with 100-fold excess S-nitroscysteine over    tetramer at pH 7.4, 25° C. for 5-20 min. After various times of    treatment, the protein was desalted over a G-25 column and treated    with dithionite. The spectra show progressive polynitrosation of Hb    with time (spectra A to F in FIG. 9C). After 5 minutes of treatment    with 100-fold excess S-nitrosocysteine, 0.09 NO groups had added per    tetramer (spectrum A of FIG. 9C); after 20 minutes, at least 4 NO    groups had added (spectrum F). At intermediate time points, 0.4 NO    groups (spectrum B), 1.58 NOs (spectrum C), 2.75 NOs (spectrum D) or    2.82 NOs had added per tetramer (spectrum E).-   D. Rat Hb was treated with 100× S-nitrosoglutathione excess over    tetramer for 3 hours at pH 7.4. The protein was then desalted by    passage through a G-25 column. A portion of the desalted protein was    treated with dithionite (spectrum B in FIG. 9D; the protein of    spectrum A was left untreated by dithionite). Spectrum B in FIG. 9D    is illustrative of a ratio of 6 RNOs/Hb.-   E. A time course experiment tracking the extent of nitrosation of    HbA₀ with time was performed (FIG. 9E). Treatment of HbA₀ was with    10× excess S-nitrosocysteine at pH 7.4, 25° C. or with 100× excess    S-nitroscysteine under the same conditions. Analysis for SNO and NO    was performed by the method of Saville and by UV spectroscopy as in    Jia, L. et al., Nature 380:221-226 (1996). Under these conditions    the heme is ultimately oxidized; the rate is time dependent.

Treatment with 10× excess S-nitrosocysteine nitrosylates only the thiolgroups of the two reactive cysteine residues of HbA₀. Inositolhexaphosphate is known to shift the allosteric equilibrium towards theT-structure (ordinarily, the deoxy form). Treatment with 100× excessnitrosates additional groups; i.e., the product has more than 2 NOgroups/tetramer.

EXAMPLE 12 Effect of SNO-Hb(FeII)O₂ on Blood Flow

SNO-Hb(FeII)O₂, having a SNO/Hb ratio of 2, was prepared (from HbA₀) byreaction with S-nitrosothiol. Rats breathing 21% O₂ were injected (time0) with Hbs prepared from HbA₀ as indicated in FIG. 10 (open circles,SNO-Hb (100 nmol/kg); filled circles, SNO-Hb (1000 nmol/kg); filledsquares, unmodified Hb (1000 nmol/kg)). Three rats were used perexperiment. Blood flow was measured in brain using the H₂ clearancemethod; microelectrodes were placed in the brain stereotactically.Concomitant PO₂ measurements revealed tissue PO₂=20. Thus, SNO-Hbimproves blood flow to the brain under normal physiological conditions,whereas native Hb decreases blood flow. NO group release is promoted bylocal tissue hypoxia.

EXAMPLE 13 Effects of SNO-Hb(FeII)O₂, SNO-Hb(FeIII) and(NO)_(x)—Hb(FeIII) on Tension of Rabbit Aorta

Hemoglobin was treated with either 1:1, 10:1 or 100:1 S-nitrosocysteineto Hb tetramer for 1 hour, processed as in Example 4. The products ofthe reactions done with 1:1 and 10:1 excess were assayed by the Savilleassay and by standard spectrophotometric methods. The product of thereaction done at the 1:1 ratio is SNO-Hb(Fe)O₂; SNO-Hb(FeIII) is foundfollowing reaction with 1:10 excess CYSNO/tetramer.

The aortic ring bioassay was performed as described in Example 4. Theproduct of the reaction in which a ratio of 100:1 CYSNO/Hb tetramer wasused, contains 2 SNOs as well as NO attached to the heme. The potency ofthe 100:1 CYSNO/Hb product is much greater than that of SNO-Hb(FeIII)and is indicative of polynitrosation (see FIG. 11).

EXAMPLE 14 Effect of Oxygenation on Partially Nitrosylated Hemoglobin

The effect of oxygenation on partially nitrosylated Hb was examined byfollowing spectral changes in the Soret region upon the addition of airto partially nitrosylated Hb. Hemoglobin A (17 μM) was deoxygenated bybubbling argon through a 1 ml solution in 100 mM phosphate (pH 7.4), for45 minutes. Nitric oxide was added by injection of 0.5 μl of a 2 mMsolution, stored under nitrogen. The final heme:NO ratio was 68:1. Thesolution was slowly aerated by sequential 50 μl injections of room air.FIG. 12 shows that the initial additions of air failed to produce a trueisobestic point, indicating changes in the concentrations of at leastthree absorbent species. Later additions of air did produce a trueisobestic point, indicative of the conversion of deoxyhemoglobin tooxyhemoglobin, with the loss of nitrosyl heme. The results show thatnitrosylated Hb is not a stable end product.

EXAMPLE 15 Conversion of Nitrosylhemoglobin to SNO-Hemoglobin

The hypothesis that the nitric oxide is transferred from the heme ironto a thiol residue, forming nitrosothiol upon oxygenation, was tested.Hemoglobin A (400 μM) was deoxygenated by bubbling argon through a 1 mlsolution in 100 mM phosphate (pH 7.4), for 45 minutes. Nitric oxide wasadded by injection of an appropriate volume of a 2 mM solution, storedunder nitrogen, to achieve different NO/Hb ratios. The solutions werethen exposed to air by vigorous vortexing in an open container. Sampleswere then analyzed by Saville assay and by chemiluminescence after UVphotolysis. Data are shown as mean±standard error (n>3). FIG. 13 showsthat S-nitrosothiol is formed in this manner, and that the efficiency ofthis reaction is greatest at high ratios of heme to nitric oxide.Amounts are highest at very high NO/Hb ratios, i.e., >2:1. This resultimplies that nitrosyl Hb entering the lung is converted into SNO-Hb, asunder physiological conditions the ratio of heme to NO is high.

EXAMPLE 16 Effects Dependent Upon Heme:NO Ratio

It was proposed that the binding of nitric oxide to the heme of the βchain was inherently unstable, and that the reason for lower yields ofSNO-Hb at higher concentrations of nitric oxide, was a loss of boundnitric oxide as a result of this instability. Hemoglobin A (17.5 μM) wasdeoxygenated by bubbling argon through a 1 ml solution in 100 mMphosphate (pH 7.4), for 45 minutes. Nitric oxide was added by sequentialinjections of an appropriate volume of a 2 mM solution, stored undernitrogen. FIG. 14A: Difference spectra of the nitric oxide hemoglobinmixture and the starting deoxyhemoglobin spectrum are shown. FIG. 14B:The peak wavelength of the difference spectra plotted against theconcentration of nitric oxide added to the solution. These data showthat addition of small amounts of nitric oxide (heme:NO ratios ofapproximately 70:1) produce predominantly nitrosylhemoglobin and someoxidized hemoglobin. However, nitric oxide additions of the order of 10μM result in the formation of oxidized hemoglobin. Heme:NO ratios atthis point are approximately 7:1. As the concentration of nitric oxideis increased by further additions of nitric oxide, the predominantspecies formed becomes nitrosylhemoglobin (heme:NO ratio 1:1). Theresults in FIGS. 14A and 14B show that under anaerobic conditions, theaddition of increasing quantities of nitric oxide to Hb results first inthe production of nitrosylhemoglobin and then oxidized Hb (metHb). Atvery high levels of nitric oxide, nitrosyl-hemoglobin is once again seenas the nitric oxide first reduces metHb to deoxyHb (producing nitrite),then binds NO. This drives the conformational change of T-structure Hbto R-structure, stabilizing the β heme-nitric oxide bond. The appearanceof oxidized Hb at heme to nitric oxide ratios of approximately 10:1indicates the decay of the heme/NO bond to produce oxidized Hb andnitric oxide anion (nitroxyl). The presence of nitric oxide anion wasconfirmed by detection of N₂O in the gas phase by gas chromatographymass spectrometry and by the production of NH₂OH.

EXAMPLE 17 Effects Upon Oxygenation of Nitrosyl-deoxyHb

Hemoglobin A (20.0 μM) was deoxygenated by bubbling argon through a 1 mlsolution in 100 mM phosphate (pH 7.4), for 45 minutes. In both FIG. 15Aand FIG. 15B, the lowest to the highest spectra indicate the sequentialadditions of air. These are difference spectra in which the pure deoxyHbspectrum occurs at zero absorbance. The peak at 419 nm is fromnitrosylhemoglobin; oxidized hemoglobin absorbs at 405 nm.

In the experiments shown in FIG. 15A, hemoglobin was graduallyoxygenated by sequential additions of 10 μl of room air by Hamiltonsyringe. Spectra are shown as difference spectra from the initialdeoxyhemoglobin spectrum. In the experiments shown in FIG. 15B, nitricoxide (1 μM) was added by injection of 0.5 μl of a 2 mM solution, storedunder nitrogen. Final heme:NO ratio was 80:1. The solution was graduallyoxygenated by sequential additions of 10 μl of room air. Spectra areshown as difference spectra from the initial deoxyhemoglobin spectrum.These data show the initial formation of a nitrosylhemoglobin peak,along with some formation of oxidized hemoglobin, which disappears afterthe addition of approximately 30 μl of air. The results indicate that asmall quantity of nitrosyl Hb is formed upon addition of low ratios ofnitric oxide to deoxy Hb, and that this nitrosyl Hb is lost uponoxygenation.

EXAMPLE 18 Role of β93Cys in Destabilizing Nitrosyl-Heme

Recombinant hemoglobins were obtained from Clara Fonticelli at theUniversity of Maryland School of Medicine. β93Ala represents a singleamino acid substitution within human hemoglobin A, whilst β93Cysrepresents a wild type control. Recombinant hemoglobin (5 μM containingeither a wild type cysteine (β93Cys) or a mutant alanine (β93Ala) atposition β93 was deoxygenated as in FIGS. 15A and 15B. Nitric oxide (1μM) was added by injection of 0.5 μl of a 2 mM solution, stored undernitrogen. The final heme:NO ratio was 20:1. The solution was graduallyoxygenated by sequential additions of 10 μl of room air. The absorptionat 418 nm of difference spectra versus initial deoxyhemoglobin spectrais shown in FIG. 16. These data indicate that within the mutant, anitrosyl adduct was formed that was not lost upon addition of room air.However, the nitrosyl adduct formed within the wild type was lost afteraddition of greater than 10 μl of room air. This shows that NO is notlost from this nitrosyl (FeII) heme in a mutant Hb that does not possessa thiol residue at position β93. Therefore, this thiol, which is inclose proximity to the heme within the R-structure, is critical fordestabilizing the heme nitric oxide bond.

EXAMPLE 19 SNO-Hb from Nitrosyl-Hb Driven by O₂

Hemoglobin A (400 μM) was prepared in a 1 ml solution, in 100 mMphosphate (pH 7.4). Nitric oxide was added by injection of anappropriate volume of a 2 mM solution, stored under nitrogen. Thesolutions were vortexed vigorously in an open container. Samples werethen analyzed by Saville assay and by chemiluminescence after UVphotolysis. The results in FIG. 17 show that S-nitrosothiol Hb can beformed from oxyHb, but that the efficiency of this formation iscritically dependent upon the ratio of heme to nitric oxide.

EXAMPLE 20 Formation of Oxidized Hb Dependent on Protein Concentration

Hemoglobin A was diluted to the concentrations indicated by thedifferent symbols in FIG. 18A and FIG. 18B, in 50 ml of 100 mM phosphatebuffer (pH 7.4). Nitric oxide was added by sequential injections of anappropriate volume of a 2 mM solution, stored under nitrogen. After eachinjection, the absorbance at 415 and 405 nm was measured. The ratio ofthese two absorbances was used to calculate the percentage content ofoxidized hemoglobin (FIG. 18A), and the absolute yield of oxidizedhemoglobin (FIG. 18B), ♦ represents 1.26 μM hemoglobin, ▪ represents 5.6μM hemoglobin, A represents 7.0 μM hemoglobin, X represents 10.3 μMhemoglobin, represents 13.3 μM hemoglobin, and ● represents 18.3 μMhemoglobin. These data show that only a small proportion of the nitricoxide added results in the formation of oxidized hemoglobin (<10%).Furthermore, this tendency to form oxidized hemoglobin is reduced athigher protein concentrations.

EXAMPLE 21 Effect of Ionic Strength and NO:Hb Ratio on Extent of MetHbFormation

We proposed that the degree of hydrogen bonding between bound oxygen andthe distal histidine was critical in determining the degree of oxidationof hemoglobin by nitric oxide. Therefore, we examined the degree ofoxidation of hemoglobin by nitric oxide in a variety of buffers. 5 ml ofphosphate buffer containing 300 μM hemoglobin A (˜95% oxyHb) was placedin a 15 ml vial. Nitric oxide was added from a stock solution, 2 mM,stored under nitrogen. Immediately after nitric oxide addition, theabsorbance at 630 nm was measured, and the concentration of oxidized(metHb) was plotted, using 4.4 as the extinction coefficient for metHbat 630 nm. Experiments were performed in 1 M, 100 mM, and 10 mM sodiumphosphate buffer (pH 7.4). The data in FIG. 19 show higher oxidizedhemoglobin formation in 1M phosphate, which is indicative of a highereffective substrate concentration, as would be predicted by phosphatedestabilization of the hydrogen bond between iron bound oxygen and thedistal histidine. At the lowest concentrations of nitric oxide added,S-nitrosothiol was formed under all conditions (approximately 5 μM).Additions of nitric oxide at concentrations of 30 μM or greater resultedin the additional formation of nitrite. The presence of 200 mM boratewithin the buffer reduced oxidized hemoglobin and nitrite formation,whilst the presence of either 200 mM or chloride increased the formationof oxidized hemoglobin and nitrite. Addition of nitric oxide tohemoglobin in 10 mM phosphate buffer at a ratio of less than 1:30(NO:Hemoglobin A) resulted in the formation of S-nitrosothiol withoutproduction of oxidized hemoglobin. S-nitrosothiol formation wasoptimized by adding the nitric oxide to hemoglobin in 10 mM phosphate,200 mM borate, pH 7.4. Therefore, the balance between oxidation andnitrosothiol formation is dependent upon the ratio of nitric oxide tohemoglobin and the buffer environment.

EXAMPLE 22 Oxygen-Dependent Vasoactivity of S-Nitrosohemoglobin:Contraction of Blood Vessels in R-Structure and Dilation in T-Structure

The details of this bioassay system have been published (Osborne, J. A.,et al., J. Clin. Invest. 83:465-473 (1989)). In brief, New Zealand Whitefemale rabbits weighing 3-4 kg were anesthetized with sodiumpentobarbital (30 mg/kg). Descending thoracic aorta were isolated, thevessels were cleaned of adherent tissue, and the endothelium was removedby gentle rubbing with a cotton-tipped applicator inserted into thelumen. The vessels were cut into 5-mm rings and mounted on stirrupsconnected to transducers (model TO3C, Grass Instruments, Quincy, Mass.)by which changes in isometric tension were recorded. Vessel rings weresuspended in 7 ml of oxygenated Kreb's buffer (pH 7.5) at 37° C. andsustained contractions were induced with 1 μM norepinephrine.

Best attempts were made to achieve equivalent baseline tone across therange of oxygen concentrations; i.e., hypoxic vessels were contractedwith excess phenylephrine. Oxygen tension was measured continuouslyusing O₂ microelectrodes (Model 733 Mini; Diamond General Co., MI)(Young, W., Stroke, 11:552-564 (1980); Heiss, W. D. and Traupett, H.,Stroke, 12:161-167 (1981); Dewhirst, M. W. et al., Cancer Res.,54:3333-3336 (1994); Kerger, H. et al., Am. J. Physiol., 268:H802-H810(1995)). Less than 1% O₂ corresponds to 6-7 torr. Hypoxic vessels werecontracted with excess phenylephrine to maintain tone. SNO-Hb[FeII]O₂(SNO-oxyHb) preparations were synthesized and quantified as in Example27; GSNO was prepared and assayed as described in Stamler, J. S. andFeelisch, M., “Preparation and Detection of S-Nitrosothiols,” pp.521-539 in Methods In Nitric Oxide Research (M. Feelisch and J. S.Stamler, eds.), John Wiley & Sons Ltd., 1996.

Hemoglobin is mainly in the R (oxy)-structure in both 95% O₂ or 21% O₂(room air) (M. F. Perutz, pp. 127-178 in Molecular Basis of BloodDiseases, G. Stammatayanopoulos, Ed. (W. B. Saunders Co., Philadelphia,1987); Voet, D. and Voet, J. G. (John Wiley & Sons Inc., New York, 1995)pp. 215-235). Hb and SNO-Hb both contract blood vessels over this rangeof O₂ concentrations. That is, their hemes sequester NO from theendothelium. The functional effects of these hemoproteins in bioassaysare not readily distinguished (FIG. 20A). Concentration-effect responsesof SNO-Hb are virtually identical to those of native Hb in 95% O₂—i.e.,in R-structure (curves are not different by ANOVA; n=12 for each datapoint). Comparable contractile effects were seen with up to 50 μMSNO-oxyHb/oxyHb—i.e., at doses where the responses had plateaued.Similar concentration-effect responses were observed in 21% O₂, underwhich condition Hb/SNO-Hb is ˜99% saturated.

On the other hand, hypoxia (<1% O₂ [˜6 mm Hg] simulating tissue PO₂)which promotes the T-structure (M. F. Perutz, pp. 127-178 in MolecularBasis of Blood Diseases, G. Stammatayanopoulos, Ed. (W. B. Saunders Co.,Philadelphia, 1987); Voet, D. and Voet, J. G. (John Wiley & Sons Inc.,New York, 1995) pp. 215-235), differentiates Hb and SNO-Hb activities:Hb strongly contracts blood vessels in T structure whereas SNO-Hb doesnot (FIG. 20B). Concentration-effect responses of SNO-Hb and Hb aresignificantly different <1% O₂ (˜6 torr), i.e. in T-structure. NativedeoxyHb is a powerful contractile agent whereas SNO-deoxyHb has a modesteffect on baseline tone. (In most experiments SNO-Hb caused a smalldegree of contraction at lower doses and initiated relaxations at thehighest dose; in some experiments (see FIG. 21C) it causeddose-dependent relaxations.) n=13 for each data point; *P<0.05;***P<0.001 by ANOVA.

SNO-Hb relaxations are enhanced by glutathione through formation ofS-nitrosoglutathione (GSNO) (FIG. 20C). The potentiation of SNO-Hbvasorelaxation by glutathione is inversely related to the PO₂ (FIG. 20C)because NO group transfer from SNO-Hb is promoted in the T-structure.Specifically, transnitrosation of glutathione by SNO-Hb—forming thevasodilator GSNO—is accelerated in T-structure (<1% O₂). Addition of 10μM glutathione to bioassay chambers potentiates the vasorelaxantresponse of SNO-Hb. The potentiation is greatest under hypoxicconditions; i.e., the curve for <1% O₂ shows a statistically significantdifference from both the 95% and 21% O₂ curves (P<0.001), which are notdifferent from one another by ANOVA (n=6 for all data points). Highconcentrations of glutathione (100 μM-1 mM) further potentiate SNO-Hbrelaxations, such that the response is virtually identical to that seenin the presence of GSNO in FIG. 20D. Glutathione at 10 μM has no effecton native Hb contractions.

In contrast, the vasorelaxant effects of S-nitrosoglutathione arelargely independent of PO₂ (FIG. 20D) and unmodified by superoxidedismutase. (Data not shown.) Concentration-effect responses ofS-nitrosoglutathione (GSNO) are largely independent of PO₂ in thephysiological range (n=6 at each data point). Results are consistentwith known resistance of GSNO to O₂/O₂ ⁻ inactivation (Gaston, B. etal., Proc. Natl. Acad. Sci. USA, 90:10957-10961 (1993)). Thus, inT-structure, relaxation by SNO overwhelms the contraction caused by NOscavenging at the heme, whereas the opposite is true in R-structure.

EXAMPLE 23 Bioactivity of Intraerythrocytic S-Nitrosohemoglobin(SNO-RBCs)

Contractile effects of RBCs are reversed by intracellular SNO-Hb in lowbut not high PO₂—i.e., under conditions that promote the T-structure.Low and high dose effects of SNO-RBCs are shown in FIGS. 21A and 21B,respectively.

Preparation of vessel rings and methods of bioassay are described inExample 22. SNO-oxyHb was synthesized and quantified as described inExample 27. Red blood cells containing SNO-Hb (SNO-RBCs) weresynthesized by treatment with tenfold excess S-nitrosocysteine overhemoglobin for 5-10 min. Under this condition, red blood cells arebright red and contain SNO-oxyHb; metHb was not detectable in theseexperiments.

Red blood cells containing SNO-Hb (SNO-RBCs) function in vessel ringbioassays like cell-free SNO-Hb. In particular, low concentrations ofSNO-RBCs (˜0.1 μM SNO-Hb) elicited modest contractile effects in 95% O₂,but not under hypoxia (FIG. 21A). In 95% O₂, both SNO-RBCs (˜0.1 μMSNO-Hb[FeII]O₂) and native RBCs produced modest contractile effects thatwere not readily distinguished. The contractions by RBCs tended to begreater under hypoxic conditions (<1% O₂), whereas those of SNO-RBCswere reversed (slight relaxant effects were seen). These O₂-dependentresponses of SNO-RBCs closely resemble those of cell-free preparations.Hemolysis was minor and could not account for the observed effects.

At higher concentrations, SNO-RBCs produced small transient relaxationsin 95% O₂ and larger sustained relaxations under hypoxia (FIG. 21B),much like cell-free SNO-Hb in the presence of glutathione. For example,SNO-RBCs (˜1 μM SNO-Hb[FeII]O₂) caused 32.5±1.2% relaxation that lasted14.5±0.7 min. in 95% O₂ versus 61±10% relaxation that lasted 23±min. in<1% O₂ (n=3−4; P<0.05). In contrast, RBCs containing no SNO-Hb producedsmall contractions (less than those of cell-free Hb) that arepotentiated by hypoxia (13±2.0% in 95% O₂ vs. 25±5% in <1% O₂; P<0.05).Hemolysis in these experiments was minor and could not account for theextent of relaxation by SNO-RBCs.

In 95% O₂; SNO-RBCs (˜1 μM SNO-Hb[FeII]O₂) produced relaxations ofaortic rings, whereas native RBCs produced slight contractions. Botheffects were more prominent at low PO₂. That is, relaxations andcontractions of intraerythrocytic SNO-Hb and Hb, respectively, weregreater and longer-lived in <1% O₂ than in 95% O₂. The O₂-dependentresponses of SNO-RBCs mimicked those of cell-free SNO-Hb in the presenceof glutathione. Hemolysis in these experiments was minor and could notaccount for the extent of relaxation by SNO-RBCs.

The normal response of systemic arteries to hypoxia is dilation andcontraction to high PO₂. The responses of vessel rings to changes in PO₂in the presence of SNO-Hb and Hb were tested (FIG. 21C). Vessel ringswere contracted with phenylephrine under hypoxic conditions (6-7 torr)and then exposed to either 1 μM Hb or SNO-Hb. Hb produced progressiveincreases in vessel tone, while SNO-Hb caused relaxations. Introductionof 95% O₂ led to rapid contractions in both cases. Thus, structuralchanges in SNO-Hb effected by PO₂ are rapidly translated intocontractions or relaxations, whereas Hb contracts vessels in both R- andT-structures. Thus, Hb opposes the physiological response and SNO-Hbpromotes it (FIG. 21C). Direct effects of O₂ on smooth muscle operate inconcert with SNO-Hb to regulate vessel tone.

EXAMPLE 24 Influence of O₂ Tension on Endogenous Levels ofS-Nitrosohemoglobin (SNO/Hb) and Nitrosyl Hemoglobin (FeNO/Hb)

Allosteric control of SNO-Hb by O₂ was assessed in vivo by perturbationof the periarteriolar oxygen gradient. In animals breathing room air(21% O₂), the precapillary resistance vessels (100-10 μm) are exposed toPO₂s as low as 10-20 torr (Duling, B. and Berne, R. M. CirculationResearch, 27:669 (1970); Popel, A. S., et al., (erratum Am. J. Physiol.26(3) pt. 2) Am. J. Physiol. 256, H921 (1989); Swain, D. P. and Pittman,R. N., Am. J. Physiol. 256, H247-H255 (1989); Buerk, D. et al.,Microvasc. Res., 45:134-148 (1993)) (confirmed here) which promotes theT-structure in Hb. Raising the inspired oxygen concentration to 100%translates to periarteriolar PO₂S only as high as 40 mm Hg (Duling, B.and Berne, R. M. Circulation Research, 27:669 (1970); Popel, A. S., etal., (erratum Am. J. Physiol. 26(3) pt. 2). Am. J. Physiol. 256, H921(1989); Swain, D. P. and Pittman, R. N. Am. J. Physiol. 256, H247-H255(1989); Buerk, D. et al., Microvasc. Res., 45:134-148 (1993)); i.e.,breathing 100% O₂ may not fully maintain the R-structure in Hb in themicrocirculation. Elimination of the periarteriolar O₂ gradient(artery-arteriole and arterial-venous difference in PO₂) is accomplishedin hyperbaric chambers by applying 3 atmospheres of absolute pressure(ATA) while breathing 100% O₂ (Tibbles, P. M. and Edelsberg, J. S.,N.E.J.M., 334:1642-1648 (1996)).

Adult male Sprague-Dawley rats (290-350 g) were anesthetized with sodiumpentobarbital (50 mg/kg IP), intubated and ventilated with a smallanimal respirator (Edco Scientific Inc., Chapel Hill, NC) at a rate andtidal volume to maintain normal values of PaCO₂ (35-45 mm Hg;PaCO₂=systemic arterial blood carbon dioxide tension). The femoral veinand artery were cannulated for infusion of drugs and for continuousmonitoring of systemic blood pressure, respectively. Aliquots ofarterial blood (200 μl) were drawn periodically to measure blood gastensions and pH (Instrumentation Laboratory Co., model 1304 blood gas/pHanalyzer). The blood was replaced intravenously with three volumes ofnormal saline. The inspired O₂ concentration was varied using premixedgases balanced with nitrogen. The tissue PO₂ was measured continuouslywith polarographic platinum microelectrodes (50 μm O.D. coated withhydrophobic gas permeable Nafion) implanted stereotaxically in both theright and left hippocampus (AP-3.4 mm, ML+2.2 mm), caudate putamennucleus and substantia nigra (see coordinates below) (Young, W., Stroke,11:552-564 (1980); Heiss, W. D. and Traupett, H., Stroke, 12:161-167(1981); Dewhirst, M. W. et al., Cancer Res., 54:3333-3336 (1994);Kerger, H. et al., Am. J. Physiol., 268:H802-H810 (1995)). The PO₂electrodes were polarized to −0.65V against a distant Ag/AgCl referencelocated on the tail and the current flow was measured using alow-impedance nA-meter. Regional arterial PO₂ was adjusted by changingthe inspired O₂ concentration and atmospheric pressure.

Polarographic hydrogen (H₂)-sensitive microelectrodes were implantedstereotaxically in the substantia nigra (AP −5.3 mm, ML −2.4 mm to thebregma, depth 3.2 mm), caudate putamen nucleus (CPN) (AP +0.8 mm, ML−2.5 mm, depth 5.2 mm) and parietal cortex, for measurement of regionalblood flow (Young, W., Stroke, 11:552-564 (1980); Heiss, W. D. andTraupett, H., Stroke, 12:161-167 (1981)). The microelectrodes were madefrom platinum wire and insulated with epoxy, with the exception of thetip (1 mm) which was coated with Nafion. For placement, the electrodeswere mounted on a micromanipulator and the rat's head was immobilized ina Kopf stereotaxic frame. H₂-sensitive electrodes were polarized to +400mV against a distant reference electrode on the tail, and thepolarographic current was measured using a low-impedance nA meter duringand after the inhalation of hydrogen gas (2.5%) for 1 min. Both thehydrogen clearance curves and voltage for oxygen measurements were madeusing PC WINDAQ (software, DI-200 AC, Dataq Instruments, Inc., Akron,Ohio). Cerebral blood flow was calculated using the initial slope method(Young, W., Stroke, 11:552-564 (1980); Heiss, W. D. and Traupett, H.,Stroke, 12:161-167 (1981)). Regional blood flow responses were monitoredfor 30 min. prior to and 30 min. following drug administration;hemoglobins were given at time 0.

Blood was drawn from indwelling catheters in the carotid artery(arterial blood that perfuses the brain) and superior vena cava/rightatrium (venous return to the heart) of 5 rats exposed first to room air(21% O₂) and then 100% O₂+3 ATA in a hyperbaric chamber. Levels ofSNO-Hb and nitrosyl Hb (Hb[Fe]NO) were determined from these samples asa measure of SNO-Hb and nitrosyl Hb (Hb[Fe]NO; FeNO/Hb in FIG. 9) inblood that perfuses the brain. The mean O₂ saturation of venous blood(room air) was 69%; of arterial blood (room air) was 93%; of venousblood (100%+3 ATA) was also 93% and of arterial blood (100%+3 ATA) was100% (FIG. 22). Numerous statistical comparisons were highlysignificant. For example, SNO-Hb venous 100% O₂+3 ATA vs. SNO-Hb venous21% O₂, P=0.004; and nitrosyl Hb venous 21% O₂ vs. arterial 21% O₂P=0.008. On the other hand, SNO-Hb and nitrosyl Hb were not different inartery 21% O₂ compared with venous 100%+3 ATA (which have identical O₂saturations), nor did the differences reach significance between venousand arterial 100% O₂+3 ATA. n=5 for all measurements.

In 21% O₂, venous blood contained mostly nitrosyl Hb, whereas arterialblood contained significant amounts of SNO-Hb (FIG. 22). On the otherhand, SNO-Hb predominated in both arterial and venous blood in 100% O₂+3ATA (FIG. 22). In hyperbaric conditions, the tissues are oxygenatedprimarily by O₂ dissolved in plasma. Physiologically circumventing theunloading of O₂ by Hb alters the endogenous SNO/nitrosyl Hb balance. Thedata show that SNO-Hb appears to form endogenously in R-structurewhereas SNO is released in the T-structure (compare venous 21% O₂(T-state) with arterial 100% O₂+3 ATA (R-state)).

This structure-function relationship in vivo is consistent with both thein vitro pharmacology and the molecular model suggesting that 1) O₂ isan allosteric effector of Hb S-nitrosylation; 2) binding of NO to hemesof Hb is favored in the T-structure; (some of the NO released duringarterial-venous (A-V) transit appears to be autocaptured at the hemes)and 3) maintaining endogenous SNO-Hb in the R-structure by eliminatingthe A-V O₂ gradient preserves levels of SNO (compare venous 100% O₂+3ATA with arterial 21% O₂). Thus, it can be predicted that SNO-Hb shouldimprove cerebral blood flow in 21% O₂, under which condition SNO isreadily released during A-V transit, but not under the hyperoxicconditions that maintain the R-structure in artery and vein.

EXAMPLE 25 O₂-Dependent Effects of SNO-Hb and Hb on Local Cerebral BloodFlow

The cerebrovascular effects of SNO-Hb were measured in adult maleSprague-Dawley rats using O₂ and H₂ (blood flow)-sensitivemicroelectrodes that were placed stereotaxically in several regions ofthe brain as for Example 24.

SNO-Hb increases blood flow under tissue hypoxia, whereas it decreasesblood flow under hyperoxia. In contrast, Hb decreases blood flowirrespective of the PO₂. Comparative effects of SNO-Hb (●) and Hb (▪) (1μmol/kg infused over 3 minutes) on local blood flow in substantia nigra(SN), caudate putamen nucleus, and parietal cortex are shown for threedifferent conditions. In 21% O₂, SNO-Hb improved blood flow in all threeregions of the brain tested, whereas native Hb decreased local bloodflow, paradoxically attenuating O₂ delivery to hypoxic tissues (FIGS.23A, 23B and 23C; all curves are highly statistically significantlydifferent from one another and from baseline by ANOVA). In ratsbreathing 100% O₂, where the periarteriolar O₂ gradient has beenessentially eliminated, the increase in flow to SNO-Hb was significantlyattenuated (i.e., only the SN increase reached statisticalsignificance), but the Hb-mediated decrease in flow was preserved (FIGS.23D, 23E and 23F; all curves remain different from one another by ANOVAto P>0.05). In 100% O₂+3 ATA, both SNO-Hb and Hb tended to decreasecerebral flow to similar extents (FIGS. 23G, 23H and 23I; curves are notdifferent by ANOVA). S-nitrosoglutathione (GSNO) increased brainperfusion in 100% O₂ and 100% O₂+3 ATA, reversing protectivevasoconstriction. Baseline blood flow was decreased by ˜10% under 100%O₂+3 ATA as compared to 100% O₂. n=7 for all data points. Values oftissue/microvascular PO₂ ranged from 19-37 mm Hg in 21% O₂; from 68-138mm Hg in 100% O₂; and from 365-538 mm Hg in 100%+3 ATA (Duke UniversityMedical Center Hyperbaric Chambers).

SNO-Hb acts like native Hb (net NO scavenger) when it is in the R(oxy)-structure and like GSNO (net NO donor) in the T (deoxy)-structure.The results are consistent with the conclusion that SNO-Hb is anitrosothiol whose vasoactivity is allosterically controlled by PO₂.

EXAMPLE 26 Hemodynamics of Cell Free and Intraerythrocytic SNO-Hb, Hband GSNO at Different O₂ Concentrations

Rats were anesthetized by intraperitoneal injection of pentobarbital,and the femoral arteries and veins accessed by local cutdown. The arterywas then cannulated and the blood pressure monitored continuously usinga P23 XL pressure transducer (Viggo Spectramed, Oxnard, Calif.) attachedto a Gould recorder. The femoral vein was used for infusion of drugs andred blood cells containing SNO-Hb (1 ml over 1 min.) and an IBM PC(WINDAQ 200, Dataq Instruments, Inc.; Akron, Ohio) was used for dataacquisition.

Drugs were infused through the femoral vein at 1 μmol/kg infused over 1minute after blood pressure had stabilized (approximately 30 min).Measurements shown (FIG. 24A) were taken at 10 min. post-infusion ofdrug. Similar responses were seen at 3 and 20 min. SNO-Hb producedsignificantly less of an increase in blood pressure than Hb (P<0.05),whereas GSNO decreased blood pressure. P<0.05 vs. SNO-Hb; *P<0.05,**P<0.01) vs. baseline blood pressure. n=5-6 for each drug.

Infusions of SNO-RBCs also lowered blood pressure consistent with aGSNO-like effect (FIG. 24B). SNO-RBCs produced dose-dependenthypotensive effects (similar to those of cell-free SNO-Hb) (P<0.001 atall points vs. baseline). The hypotensive effects of SNO-RBCs werepotentiated by pre-administration of the NO synthase inhibitorN^(G)-monomethyl-L-arginine (L-NMMA; 50 mg/kg). n=8 for each data point.Curves different by ANOVA (P<0.01), *P<0.05 vs. L-NMMA. The amount ofhemolysis in these experiments was trivial. Infusion of the hemolysatehad no effect on blood pressure.

NO synthase inhibition increases tissue O₂ consumption by relieving theinhibition of mitochondrial respiration produced by NO in the tissues(King, C. E. et al., J. Appl. Physiol., 76(3):1166-1171 (1994); Shen, W.et al., Circulation, 92:3505-3512 (1995); Kobzik, L. et al., Biochem.Biophys. Res. Comm., 211(2):375-381 (1995)). This should, in turn,increase the periarteriolar O₂ gradient which might explain some of thepotentiation. However, other factors, such as a change in tone ordistribution of blood flow imposed by L-NMMA, may well contribute. Theeffects of SNO-Hb on blood pressure are consistent with SNO beingreleased in resistance arterioles to compensate for NO scavenging at theheme iron.

EXAMPLE 27 Synthesis of S-Nitroso-Oxyhemoglobin (SNO-Hb[Fe(II)]O₂) andSNO-metHb (SNO-Hb[FeIII])

Hemoglobin (Hb)A_(o) was purified from human red blood cells aspreviously described (Kilbourn, R. G. et al., Biochem. Biophys. Res.Commun., 199:155-162 (1994)). HbA_(o) was dialyzed against 2% borate,0.5 mM EDTA (pH 9.2) at 4° C. for 12-16 hours. The oxyHb concentrationwas determined based on the optical absorbance at 577 nm (i.e., usingthe millimolar extinction coefficient 14.6).

Hb was reacted with 10-fold molar excess S-nitrosocysteine (CYSNO) whichwas synthesized with modification of standard procedure (see, forexample, Stamler, J. S. and Feelisch, M., “Preparation and Detection ofS-Nitrosothiols,” pp. 521-539 in Methods In Nitric Oxide Research (M.Feelisch and J. S. Stamler, eds.), John Wiley & Sons Ltd., 1996) asfollows. L-cysteine hydrochloride (1.1 M) dissolved in 0.5 N HCl/0.5 mMEDTA was reacted with an equal volume of 1 M NaNO₂ (sodium nitrite)dissolved in water, to form CYSNO (the ratio of cysteine to nitriteinfluences the SNO-Hb product and activity profile). The concentrationof CYSNO was then adjusted by dilution in 200 mM phosphate buffer (PBS),pH 8.0, to yield a working CYSNO solution (pH 6-7). This was thenreacted at room temperature with a 20-fold volume excess of Hb (inborate, pH 9.2), resulting in a final 10-fold molar excess of CYSNO overHb (ratio influences product critically). Following the incubation(periods determined by the desired synthetic preparation; i.e., adesired ratio of SNO/tetramer; desired met- to oxy- to nitrosyl-Hbratios; polynitrosated or non-polynitrosated; for example, 10 min.preferred time for SNO-oxyHb with 2 SNO per tetramer; see below), thereaction mixture was rapidly added to a column of fine Sephadex G-25 (20to 30-fold volume excess over the reaction mixture) preequilibrated with100 mM PBS pH 7.4, 0.5 mM EDTA. Typically, a 150 μl sample of themixture was added to a 4.5 ml column measuring 12 mm (inner diameter).The column was then centrifuged at 1200 g for 60 seconds and theeffluent collected in a 1.5 ml airtight plastic vial that wassubsequently kept on ice and protected from light.

Spectrophotometric determination of total Hb and S-nitrosothiol (SNO)concentration was determined by Saville assay (especially modified fromstandard protocol (see Materials and Methods section of Exemplification;see also, for example, Stamler, J. S. and Feelisch, M., “Preparation andDetection of S-Nitrosothiols,” pp. 521-539 in Methods In Nitric OxideResearch (M. Feelisch and J. S. Stamler, eds.) John Wiley & Sons Ltd.,1996; Saville, B., Analyst, 83:670-672 (1958)) by keeping the Hb in thesample to 5 μl per 50 μl sample volume added to assay, adding 0.1-1%Tween as necessary, and by correcting for nitrosyl Hb content convertedto nitrite).

Incubations of CYSNO with HbA resulted in different synthetic products(and different activities) over time. For example, with 10 minincubations, the Hb preparation contains 1.857±0.058 SNO groups pertetramer, and is approximately 12-15% metHb and 1-3%nitrosyl(FeII)-hemoglobin. (Capillary electrophoretic analysis actuallyreveals a mixture of three protein peaks.) MetHb was then reduced(lowered from 13% to 2% with 100-fold excess NaCNBH₃ (dissolved in PBS,pH 8.0) under anaerobic conditions (achieved by purging with argon gasfor a minimum of 10 minutes) for 5 minutes. (Lower concentrations ofNaCNBH₃ or treatment of the samples under aerobic conditions were noteffective in lowering the metHb concentration, and alternative measuresto reduce the heme result in SNO reduction.) The resulting mixture wasrapidly added to a column of fine Sephadex G-25 (20 to 30-fold volumeexcess) preequilibrated with 100 mM PBS pH 7.4, 0.5 mM EDTA. The finalS-nitrosothiol/Hb tetramer ratios were not significantly different fromthose measured in samples degassed and treated with PBS only: losses inSNO/Hb ratio relative to the starting ratios were consistent with theexpected time-dependent decay of SNO-deoxyHb and could be reduced to aninsignificant loss by taking preparitive time into consideration.NaCNBH₃ treatment of a sample with a mean SNO/Hb ratio of ˜1 decreasedthe metHb content from 5.6% to 0.63%. Nitrosyl(FeII)-hemoglobin andmetHb contamination of SNO-oxyHb preparations of ˜2% are acceptable,inasmuch as they do not seem to alter bioactivity of SNO-oxyHb, andenable O₂ binding measurements, that is, P₅₀ determinations (so calledSNO-Hb[Fe(II)]O₂). By the same token, bioactivity can be modified andvaried by controlling the proportion of SNO-metHb (high and low spin)and nitrosyl(FeII)-hemoglobin in the preparation. The spin state ofmetHb can be controlled by the heme ligand: cyan-metHb is low spin andaquo-met Hb (H₂O bound as ligand) is high spin; nitrosyl Hb ratios aremade depending on the desired result (see, inter alia, Example 16). Highyield SNO-metHb, SNO-nitrosyl(FeII)-hemoglobin (or SNO-carboxyl Hb) canbe formed by using the heme-liganded protein as starting material.Carboxyl Hb can be made by gassing with CO under anaerobic conditions.HbCO can then be used as starting material; likewise, variouscombinations of Hb[FeNO] [FeCO] can be used as starting material.

Equivalents

Those skilled in the art will know, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and all otherequivalents are intended to be encompassed by the following claims.

1. A method for treating a disorder resulting from platelet activationor adherence in an animal or human, comprising administering to theanimal of human a composition comprising nitrosylhemoglobin in atherapeutically effective amount, wherein the disorder is selected fromthe group consisting of: myocardial infarction, pulmonarythromboembolism, cerebral thromboembolism, thrombophlebitis, sepsis andunstable angina.